Methods of reducing expression of x-inactivation escapee genes and autosomal genes

ABSTRACT

Inhibitory nucleic acids, e.g., antisense oligonucleotides (ASO) against PAR-TERRA RNA or other chromosome-specific TERRA transcripts (i.e., inclusive of chromosome-specific subtelomeric sequences), and methods of use thereof to downregulate expression of escapee genes on the inactive X chromosome, expression from the active X chromosome, subtelomeric autosomal loci (e.g., FSHD locus), or expression of autosomal genes involved in growth control and apoptosis, e.g., in cells and subjects with supernumerary X chromosomes and/or cancer and other human diseases.

CLAIM OF PRIORITY

This application claims the benefit of U.S. Provisional PatentApplication Ser. No. 62/261,698, filed on Dec. 1, 2015. The entirecontents of the foregoing are hereby incorporated by reference.

FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

This invention was made with Government support under Grant No.R01-GM58839 awarded by the National Institutes of Health. The Governmenthas certain rights in the invention.

TECHNICAL FIELD

Described herein are inhibitory nucleic acids, e.g., antisenseoligonucleotides (ASO) against PAR-TERRA RNA and TERRA of autosomalorigin, and methods of use thereof to downregulate: (i) expression ofescapee genes on the inactive X chromosome (Xi), e.g., in cells andsubjects with supernumerary X chromosomes, (ii) expression from theactive X chromosome (Xa), (iii) expression a network of autosomal genesinvolved in growth control and apoptosis, and (iv) expression of thegenetic locus associated with FSHD (facioscapulohumeral musculardystrophy) and other subtelomeric autosomal genes.

BACKGROUND

The mammalian genome is ubiquitously transcribed and the ends oftelomeres are no exception. In spite of their heterochromaticproperties, telomeric ends actively synthesize a heterogeneouspopulation of long noncoding RNAs dubbed “TERRA” (Azzalin et al., 2007;Schoeflner and Blasco, 2007; Zhang et al., 2009). TERRA transcriptsrange in size from 100 bases up to >9 kb and contain the canonicaltelomeric repeat sequence, UUAGGG, as well as sequences unique to thesub-telomeric region of each chromosome. The function of TERRA hasgenerated major interest in light of its association with humandiseases, such as cancer and the ICF syndrome (immunodeficiency,centromere instability, and facial anomalies) (Maicher et al., 2012;Azzalin and Lingner, 2015). Elegant studies have pointed to a number oftelomere-associated functions. Telomeres are well-defined nucleoproteincomplexes that cap the physical ends of linear chromosomes and protectthem from unprogrammed shortening and genetic rearrangements (Blackburnet al., 2006; Sfeir and de Lange, 2012; Bernardes de Jesus and Blasco,2013; Doksani and de Lange, 2014; Azzalin and Lingner, 2015). Thereverse transcriptase activity of the RNA-containing telomerase complexenables regeneration of chromosomal ends that are lost with every DNAreplication (Lingner et al., 1997). However, TERRA's activity does notappear to be directly related to telomerase activity (Schoeftner andBlasco, 2007; Redon et al., 2010; Redon et al., 2013). Rather, TERRAseems to keep telomere length in check (Sandell et al., 1994; Luke etal., 2008; Maicher et al., 2012; Pfeiffer and Lingner, 2012; Pfeiffer etal., 2013; Wang et al., 2015), regulate recombination (Balk et al.,2013; de Silanes et al., 2014; Yu et al., 2014), and serve as a scaffoldfor recruitment of HP1, histone methyltransferases, and shelterins totelomeric heterochromatin (Deng et al., 2009). Thus, TERRA is anintegral part of the telomeric architecture.

Cytological studies indicate that only about half of detectable TERRAtranscripts are localized to telomeres (Le et al., 2013). The remaininghalf is presumed to be “free” in the nucleoplasm. Nevertheless,investigation into TERRA function has focused almost exclusively ontelomeres, though early observations noted a large cluster of TERRAtranscripts near the inactive X-chromosome (Xi) of somatic female cells(Schoeftner and Blasco, 2008; Zhang et al., 2009). TERRA RNA is alsoconcentrated next to the Y-chromosome (Zhang et al., 2009).

SUMMARY

Telomeric repeat-containing RNAs (TERRA) are highly conserved longnon-coding RNAs transcribed from telomeric ends of eukaryoticchromosomes. TERRA has so far only been ascribed function in telomerebiology. Genome-wide binding sites for TERRA have now been identified,and show that TERRA localization is not cis-limited, nor is TERRAfunction confined to telomeres. Transcriptomic analysis shows that TERRAdepletion results in dysregulation of TERRA target genes. Describedherein is a subclass of TERRA transcripts specific to the sexchromosomes. Dubbed PAR-TERRA, these transcripts originate within thepseudoautosomal region (PAR) and mediate two special sex-linkedprocesses. First, in somatic cells, PAR-TERRA prevents spreading of XistRNA away into genes that escape silencing on the inactive X (Xi).PAR-TERRA renders X-linked escapee genes immune to Xist RNA. DepletingPAR-TERRA leads to downregulation of escapees. We also show thatdepleting PAR-TERRA reduces expression of the Xa gene and various targetautosomal genes, especially those involved in apoptosis and cell cycleregulation. Thus, the methods can also be applied to downregulate anetwork of autosomal genes involved in growth control and apoptosis.Provided herein is evidence that PAR-TERRA sets up a specializedprivileged compartment that aids in boosting transcriptional activityspecific genes across the genome. Thus, PAR-TERRA may be targeted toturn down (i) expression of escapee genes on the inactive X chromosome(Xi), e.g., in cells and subjects with supernumerary X chromosomes, (ii)expression from the active X chromosome (Xa), or (iii) expression anetwork of autosomal genes involved in growth control and apoptosis. Thepresent methods include using inhibitory nucleic acids, e.g., antisenseoligonucleotides (ASO) against PAR-TERRA RNA to downregulate expressionof these classes of genes.

Thus, provided herein are isolated inhibitory nucleic acids targetingPAR-TERRA, preferably wherein the inhibitory nucleic acid is modified,and compositions comprising the isolated nucleic acids.

Also provided are methods for decreasing expression of an Xi escapeegene in a cell, preferably a cell of a subject have a supernumerary Xchromosome. The methods include administering to the cell an inhibitorynucleic acid targeting PAR-TERRA, preferably wherein the inhibitorynucleic acid is modified.

Further, provided are methods for decreasing expression of Xa genes in acell, preferably a cell of a subject having a supernumerary Xchromosome. The methods include administering to the cell an inhibitorynucleic acid targeting PAR-TERRA, preferably wherein the inhibitorynucleic acid is modified.

Also provided are methods for treating a subject who has a disorder ofsex chromosome aneuploidy associated with a supernumerary X chromosome.The methods include administering to the subject an inhibitory nucleicacid targeting PAR-TERRA, preferably wherein the inhibitory nucleic acidis modified.

Also provided is a composition described herein, e.g., comprising aninhibitory nucleic acid targeting PAR-TERRA, for treating a subject whohas a disorder of sex chromosome aneuploidy associated with asupernumerary X chromosome.

In some embodiments described herein, the subject has 46,XY, 47,XXY,48,XXYY, 48,XXXY, 47,XXX, 48,XXXX or 49,XXXXX aneuploidy.

In some embodiments described herein, the cell is from a subject who has46,XY, 47,XXY, 48,XXYY, 48,XXXY, 47,XXX, 48,XXXX or 49,XXXXX aneuploidy.

Also provided are methods for decreasing expression of X-linked,autosomal growth control or apoptosis genes, and sub-telomeric autosomalgenes in a cell (e.g., out D4Z4, DUX4, FRG1, and FRG2 for FSHD, fromChr4). The methods include administering to the cell an inhibitorynucleic acid targeting PAR-TERRA, PAR, or TERRA, preferably wherein theinhibitory nucleic acid is modified.

In addition, provided are methods for decreasing expression of autosomalgenes in a cell. The methods include administering to the cell aninhibitory nucleic acid targeting PAR-TERRA or an autosome-specificTERRA (e.g., TERRA species originating with the subtelomeric region ofan autosome and comprising autosome-specific 5′ sequences), preferablywherein the inhibitory nucleic acid is modified. In some embodiments,the inhibitory nucleic acid targets Chr4-specific TERRA. In someembodiments, expression of FRG1, FRG2, DUX4, and the long noncoding RNAsof forward and reverse orientations from the macrosatellite repeat, D4Z4is decreased. In some embodiments, the cell is from or in a subjectionwho has facioscapulohumeral muscular dystrophy (FSHD). Thus, in aspecific example the methods include targeting the Chr4 regionassociated with facioscapulohumeral muscular dystrophy (FSHD), which islocated in the subtelomeric region of human Chr4 and contains codinggenes FRG1, FRG2, DUX4, and the long noncoding RNAs of forward andreverse orientations from the macrosatellite repeat, D4Z4. FSHD iscaused by ectopic expression of these genes when the D4Z4 repeatcontracts and becomes “activated”. Thus, PAR-TERRA or Chr4-specificTERRA can be targeted to downregulated the associated subtelomericgenes. Other subtelomeric genes, e.g., as shown in FIG. 4A,B, from 5chromosomes are shown (Chr1,3,8,18,19), can also be targeted.

In some embodiments described herein, the inhibitory nucleic acid doesnot comprise three or more consecutive guanosine nucleotides or does notcomprise four or more consecutive guanosine nucleotides.

In some embodiments described herein, the inhibitory nucleic acid is 8to 30 nucleotides in length.

In some embodiments described herein, at least one nucleotide of theinhibitory nucleic acid is a nucleotide analogue.

In some embodiments described herein, at least one nucleotide of theinhibitory nucleic acid comprises a 2′O-methyl, e.g., wherein eachnucleotide of the inhibitory nucleic acid comprises a 2′O-methyl.

In some embodiments described herein, the inhibitory nucleic acidcomprises at least one ribonucleotide, at least one deoxyribonucleotide,or at least one bridged nucleotide.

In some embodiments described herein, the bridged nucleotide is a LNAnucleotide, a cEt nucleotide or a ENA modified nucleotide.

In some embodiments described herein, each nucleotide of the inhibitorynucleic acid is a LNA nucleotide.

In some embodiments described herein, one or more of the nucleotides ofthe inhibitory nucleic acid comprise 2′-fluoro-deoxyribonucleotidesand/or 2′-O-methyl nucleotides.

In some embodiments described herein, one or more of the nucleotides ofthe inhibitory nucleic acid comprise one of both of ENA nucleotideanalogues or LNA nucleotides.

In some embodiments described herein, the nucleotides of the inhibitorynucleic acid comprise comprising phosphorothioate internucleotidelinkages between at least two nucleotides, or between all nucleotides.

In some embodiments described herein, the inhibitory nucleic acid is agapmer or a mixmer.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Methods and materials aredescribed herein for use in the present invention; other, suitablemethods and materials known in the art can also be used. The materials,methods, and examples are illustrative only and not intended to belimiting. All publications, patent applications, patents, sequences,database entries, and other references mentioned herein are incorporatedby reference in their entirety. In case of conflict, the presentspecification, including definitions, will control.

Other features and advantages of the invention will be apparent from thefollowing detailed description and figures, and from the claims.

REFERENCE TO SEQUENCE LISTING

This application includes a sequence listing submitted herewith inelectronic format. The entire content of this files is herebyincorporated by reference.

DESCRIPTION OF DRAWINGS

FIGS. 1A-H. Telomeric RNAs produced by the sex chromosomes

A. TERRA RNA FISH followed by PAR DNA FISH using P34567 probes, whichare subsets of BAC RP24-500I4 DNA. Higher exposure of TERRA RNA FISHrevealed that multiple TERRA foci were sparely distributed across thenucleus in mES cells. DAPI detects nuclear DNA.

B. Map of the PAR and relative positions of BAC clones, RP24-143B12 andRP24-500I4. Locations of internal (TTAGGG) repeats shown in red. Dottedpurple lines, incompletely assembled regions.

C. TERRA RNA FISH followed by PAR DNA FISH (n=204) using P34567 probesin ES cells. Lower exposure of TERRA RNA FISH showed that the dominantTERRA foci were colocalized with PAR DNA. 80-90% of TERRA signalslocalized to ChrX and Y.

D. Percent colocalization of PAR and TERRA signals. n=139 (male); 209(female).

E. Two color RNA FISH detecting TERRA (Alexa488, green) and PARtranscripts (Cy3, red) in ES cells. Nick-translated BAC DNA was used todetect PAR RNA.

F. Top panel: Map of sub-BAC probes and PCR amplicons. Left panel:Northern blot analysis of PAR-TERRA RNA using either TERRA or 36K oligoprobes in ES cells on differentiation days 0-12, as indicated. GAPDH,loading control. Right panel: Primer extension using an antisense TERRAoligo probe with PCR amplification using PAR-specific primer pairslocated at 33, 36, and 39 k (kb) from the end of BAC RP24-500I4TERRA. +,with RT; −, without RT.

G. Northern blot analysis of PAR-TERRA in ES cells using TERRA-specificor PAR-specific oligo probes, as shown in panel F.

H. Higher exposure RNA FISH indicating colocalization of TERRA and PARsignals at both large and small foci in ES cells. Three-color RNA FISH(upper panel): TERRA oligo probe; PAR-specific probes, 47k and 22k.Two-color RNA FISH (lower panel): TERRA oligo probe; PAR specific probe,31k. DAPI was used for nuclear staining. Right graph, quantitation ofcolocalization.

FIGS. 2A-I. Mapping genomic PAR-TERRA binding sites by ChIRT-seq

A. To capture the PAR-TERRA transcripts, five DNA oligo probes wereused: 22k, 31k, 34k, 36k, and 47k. Each probe has multiple alignments tothe RP24-500I4 BAC DNA around the telomeric repeats.

B. RNA slotblot analysis showing that TERRA-AS and PAR-31-AS probesspecifically captured TERRA RNA by ChIRT. Total RNA was extracted frombeads after ChIRT hybridization without RNase H elution.

C. Quantitative PCR showing the enrichment of PAR DNA in TERRA-AS ChIRTand PAR ChIRT, but not TERRA-S ChIRT in ES cells. TERRA-AS ChIRT usedantisense DNA oligos against TERRA, TERRA-S ChIRT used TERRA senseprobes as a control, and PAR ChIRT used PAR probes for the PARtranscripts.

D. Enrichment of PAR DNA following TERRA ChIRT was observed only whenelution was performed with RNaseH. Enrichment was abolished in the RNaseA pre-treated control (Pre-RNaseA).

E. Table of ChIRT results indicating the number of PAR and TERRA bindingsites in ES cells on different days of differentiation and in MEFs.Different normalization methods produced similar results, as shown.

F. Scatterplot analysis comparing log2 coverages of TERRA and PAR ChIRTin indicated samples. Pearson's r shown. ChIRT results were normalizedto input unless otherwise indicated.

G. TERRA ChIRT-seq showed enrichment for telomeric repeats DNA in femaleES cells. Samples captured by TERRA-AS or TERRA-S. No-RNAseH for theTERRA-AS capture is also shown as a control.

H. Pie charts show relative representation of various genomic regions inTERRA (top) and PAR (bottom) ChIRT-seq experiments in female ES cells.

I. CEAS analysis shows significant over-representation of introns andnoncoding regulatory regions. Exons are under-represented. ***, P<0.001(one-sided binomial test). The genome reference was obtained from theChIRT-seq input.

FIGS. 3A-D. X-linked PAR-TERRA RNA binds in cis and in trans to multiplechromosomes.

A. ChIRT-seq tracks showing PAR-TERRA enrichment at the ends of variouschromosomes in female ES cells (top) and MEFs (bottom). TERRA ChIPT-seqdata was normalized to input (TERRA/input), no-RNase H control (TERRA/noRNase H), or the sense control (TERRA/sense).

B. Magnified views of the female ES PAR-TERRA ChIRT-seq results for thepseudoautosomal regions of ChrX and ChrY.

C. Female ES ChIRT-seq tracks showing PAR-TERRA enrichment on multipleautosomes. Red bars, internal TTAGGG repeats. Grey bars, sequence gaps.

D. ChIRT-seq tracks of female ES cells showing PAR-TERRA binding tonon-telomeric autosomal regions.

FIGS. 4A-G. Transcriptome analysis of PAR-TERRA-depleted cells.

A. Northern blot analysis of TERRA RNA shows depletion by treatment withgapmer LNA against TERRA or PAR in ES cells. Control, scramble LNAgapmer (Scr KD).

B. RNA FISH detecting TERRA (Alexa-488, green), or PAR (Cy5, cyan blue)after LNA knockdown in ES cells.

C. Venn diagram of genes affected by TERRA versus PAR KD. Number ofgenes in each circle and overlapped region is indicated. 56 genes areshared between TERRA and PAR KD in female ES cells; 36 in MEF.

D. Heatmap of differentially expressed genes in TERRA KD, PAR, and ScrKD. 56 shared genes were examined for ES cells; 36 shared genes for MEF.Scale in Log10 FPKM.

E. Heatmap of differentially expressed genes in TERRA KD, PAR KD, andScr KD ES cells or MEFs. 8 genes were shared in both ES cells and MEFs.

F. Probability density function for the 565 genes with and 14,724 geneswithout PAR-TERRA binding sites in the structural gene ±10Kb of flankingsequence, with respect to their likehood of changing gene expressionfollowing PAR-TERRA KD. Log2 fold-change (AFPKM) is plotted. AfterPAR-TERRA KD, there is a net downregulation for the group of genes withPAR-TERRA binding sites. Kolmogorov-Smirnoff (KS) test, P<0.0001.

G. Cumulative fraction that genes with or without PAR-TERRA sites wouldbe up- or down-regulated following PAR or TERRA KD, as indicated.P-values determined by X² analyses.

FIGS. 5A-F. PAR-TERRA protects escapees and genes of thesubtelomeric/pseudoautosomal regions from silencing.

A. RNA-seq shows downregulation of subtelomeric genes following TERRA KDin ES cells. ChIRT-seq and post-KD RNA-seq tracks are shown.

B. RT-qPCR confirms that Tmx3 and Wls are downregulated upon TERRA KD inMEFs.

C. Whole-ChrX view of PAR-TERRA binding sites. Two regions (boxes) showhigh-level binding. Escapee genes shown below the chart.

D. Table showing the numbers of total and ChrX PAR-TERRA binding sitesin female ES cells and in MEFs.

E. Probability density functions for escapees (n=15), Xi genes (n=438)subject to XCI. Escapee genes have higher PAR-TERRA binding densitiesrelative to genes subject to XCI (P<0.001 for TERRA density, P<0.05 forPAR density, KS test).

F. RT-qPCR of pseudoautosomal genes following PAR or TERRA KD. P-valuesdetermined by the Student t-test.

FIGS. 6A-I. TERRAs regulate the gene expression on the PAR

A. Dynamics of Xist RNA spread following PAR-TERRA KD in female MEFs.Shown are tracks for Xist CHART-seq after Scr, TERRA, or PAR KD, andtracks for PAR-TERRA ChIRT-seq. Yellow-shaded region corresponds to thePAR-TERRA and Xist boundaries within Mid1.

B. Metagene analysis of PAR-TERRA density across XCI-repressed (n=438)and escapee genes (n=15). Relative PAR-TERRA density from PAR or TERRAChIRT in MEFs was produced by CEAS analysis.

C. Scatterplot analysis comparing Xist coverage (log2 scale) inPAR-TERRA KD female MEFs relative to Scr KD on ChrX. The transcriptomicprofiles are highly similar (Pearson's r>0.90). Outliers (dots) map tothe “borders” of pseudoautosomal genes. Xist coverage files werenormalized to the corresponding ChrX median values, and individual dotsin the scatterplot represents an average of two biological replicates.

D. Metagene analysis of Xist density across XCI-repressed (n=438) andescapee genes (n=15) after TERRA KD or Scr KD in MEFs.

E. RNA FISH detecting TERRA (Alexa-488) and Xist (Cy3, red) in MEFscells. 87% show colocalization (n=139).

F. 3D DNA FISH to determine the colocalization frequency of PAR (Cy3),the Xic (Ftx-Jpx probe; Cy5) and Hprt (FITC). A colocalization event isscored when two signals show overlapped pixels in 3D space. N=276nuclei. P, determined by two-tailed Fisher's exact test.

G. 3D DNA FISH to determine the frequency of PAR-Xic colocalizationafter PAR-TERRA KD. A colocalization event is scored when two signalsshow overlapped pixels. N=256-272 nuclei. P, determined by two-tailedFisher's exact test.

H. 2D model: PAR-TERRA protects escapees from Xist silencing by settingup a privileged compartment and walling off Xist at the 5′ end ofescapee genes. When PAR-TERRA is depleted, Xist spreads into theprivileged compartment.

I. 3D Model: PAR-TERRA as an organizing center. PAR-TERRA forms aprivileged nuclear compartment next to the Xist cloud. The Xi ispartitioned spatially into a silent domain and an active domain forescapees.

FIGS. 7A-I. TERRAs regulate Xic pairing in mES cells

A. PAR-TERRA ChIRT-seq tracks of the Xic pairing center (red bar) inMEFs and in ES cells on d0, d3, and d7 of differentiation. Noteprominent ES-specific PAR-TERRA peaks at the pairing center.

B. Cumulative frequency curves of inter-allelic differences measuredbetween Xic-Xic, telomere-telomere, and Hprt-Hprt (bottom). Measurementswere taken on DNA FISH experiments (representative DNA FISH image isshown) which detected Xic (Xist), TeloX (RP23-461E16, ChrX telomericBAC), and Hprt (Cy5). ES cells on d0 and d4 shown. Normalized distance(ND)=distance/d, where d=2 X (nuclear area/π)0.5. ND 0.0-0.2 are shown.n=109-120. P values were determined using the KS test.

C. Cumulative frequency curves for inter-allelic telomeric distances forChrX (TeloX) or Chr2 (Telo2) on day 4 of ES differentiation. n=120-158.P values were determined using the KS test.

D. PAR-to-PAR pairing during female and male ES cell differentiation.n=246-385. P values were determined using the KS test.

E. Cumulative frequency of paired PAR DNA (TERRA RNA signals) in male EScells on d0 versus d4. DNA FISH shows that, on d4 of differentiation,the PAR's of ChrX and ChrY were frequently colocalized (one dot) or veryclose in 3D space (2 neighboring dots of <0.2 ND). N=149 (d0); 176 (d4).P value was determined using the KS test.

F. Cumulative frequency curves show that TERRA knockdown disruptedtelomeric pairing in both female and male ES cells at 6 hrpost-transfection at d4 of differentiation. P values were determinedusing the KS test. n=235-336.

G. Cumulative frequencty curve shows that TERRA knockdown disruptedXic-Xic pairing in female ES cells at 3 hr post-trasfection on d4 ofdifferentiation. P=0.001 (KS test). n=326-377.

H. Cumulative frequency curves indicate an increase in Xic-telodistances after 3 hours of TERRA KD in d4 female ES cells. P=0.009 (KStest). n=174-214.

I. Model: Without wishing to be bound by theory, it is believed thatPAR-TERRA forms an organizing center to facilitate X-X pairing. (1)Prior to cell differentiation, the two female X-chromosomes areseparated. (2) During early cell differentiation, trans-interactionsbetween two telomeres bring the sex chromosomes in close promixity. (3)PAR-TERRA also drives the intra-chromosomal interactions between the Xicand the telomere in cis. (4) These events bring the Xic pairing centerto the same juxta-telomeric compartment, accelerating the homologysearch between the two Xic pairing centers by the reduced effectivesearch space. The pairing event induces initiation of XCI in femalecells. In male cells, the telomeric pairing interaction also occurs, butis not followed by Xic pairing; thus, XCI is not initiated.

FIGS. 8A-E. Cytological analysis of PAR-TERRA transcripts. This figurerelates to FIGS. 1A-H.

A. DNA FISH detecting PAR DNA using P34568 sub-probes of BAC RP24-500I4DNA (Cy3, red), and X chromosomes (FITC labeled X painting probes,green) on metaphase spread in female ES cells. P345678 probes mark onthe end of X chromosomes.

B. Electrophoresis of PCR products amplified from BAC RP24-500I4 DNA.The pools of P3, P4, P5, P6, and P8 PCR produces were used forgenerating P34568 sub-probes to detect PAR DNA in DNA FISH experiments.

C. RNA FISH detecting TERRA RNA in various human (lower panel) and mouse(upper panel) cell lines.

D. PAR-TERRA RNA is localized next to the Xist cloud. RNA FISH detectingTERRA (Alexa-488, green), 14-31k (Cy5, cyan blue), and Xist (Cy3, red)in MEFs (upper panel). Image of the overexposed TERRA foci (green) todisplay moderate intensity of TERRA foci was shown in the lower panel.False color for I4-31k (red, lower panel).

E. RNA FISH detecting TERRA (Cy5, cyan blue), Xist (FITC, green), thePAR transcripts with DNA oligo probes: I4-47k (Alexa-488, green) andI4-22k (Cy3, red) in MEFs (hybrid strain cas/mus, Xist cloudsspecifically on mus alleles), female ES cells (hybrid strand, cas/mus)and male ES cells (mus/mus). I4-22k probes only mark on cas alleles.

FIGS. 9A-C. ChIRT-seq statistics. This figure relates to FIG. 2.

A. Quantitative PCR showing the enrichment of PAR DNA following ChIRTusing oligo probes TERRA-AS (AS) that targets to TERRA transcripts orsense probes. Various detergents (0.1% NP40, or 0.1% SDS, or 0.1%N-lauryol Sarcosine) were added separately during the final DNA elution.NP40 retains RNase H activity better than other detergents in ChIRTelution.

B. Scatterplot comparing log2 coverages of biological replicates for PARand TERRA ChIRT-seq analysis in ES cells. Pearson's r shown. Replicate 1(Rep1) was normalized with input. Replicate 2 (Rep2) was normalized toRNaseA pre-treated control.

C. Read statistics for two biological replicates of the PAR-TERRAChIRT-seq analysis.

FIG. 10. PAR-TERRA RNA binds subtelomeric regions of select autosomes.

This figure relates to FIG. 3. ChIRT-seq tracks showing PAR-TERRAenrichment at the subtelomeric regions of Chr19, 8, and 16 in ES cells.

FIGS. 11A-C. PAR-TERRA knockdown by LNA gapmers. This figure relates toFIG. 4.

A. LNA gapmers efficiently knocked down PAR-TERRA in ES cells after 1 to48 hours.

B. RNA FISH detecting TERRA (Alexa-488, green) after LNA transfection atvarious time points in ES cells.

C. As shown by scatterplots comparing log2 FPKM values, there is goodcorrelation between biological replicates of RNA-seq biologicalreplicates after PAR-TERRA KD in ES cells and MEFs. Pearson's r asshown.

FIGS. 12A-B. Analysis of gene expression following PAR-TERRA knockdown.This figure relates to FIG. 5.

A. ChIRP-seq tracks (red) for PAR-TERRA binding sites neardifferentially downregulated genes after PAR-TERRA KD in ES cells.RNA-seq coverage are FPM-normalized and tracks are shown in blue.

B. ChIRP-seq tracks (red) for PAR-TERRA binding sites neardifferentially upregulated genes after PAR-TERRA KD in ES cells. RNA-seqcoverage are FPM-normalized and tracks are shown in blue.

FIGS. 13A-B. Telomeric pairing analysis: Whole distributions ofinter-PAR distances. This figure relates to FIG. 7.

A. Distributions of PAR-PAR distances in female ES cells on days 0, 4,and 8 of differentiation. Normalized distance (ND)=PAR-PAR distance/d,where d=2 X (nuclear area/π)0.5.

B. Distributions of PAR-PAR distances in male ES cells on days 0, 4, and8 of differentiation.

DETAILED DESCRIPTION

XCI is an epigenetic pathway that results in silencing of oneX-chromosome in the female cell to compensate for unequal X-chromosomenumber between male (XY) and female (XX) cells (Starmer and Magnuson,2009; Lee, 2011; Wutz, 2011; Disteche, 2012). The pathway is controlledby long noncoding RNAs (lncRNA) of the X-inactivation center (Xic).During early development and as recapitulated by differentiatingembryonic stem (ES) cells, the X-to-autosome ratio is assessed and theXCI pathway is induced only when there is more than one X-chromosome ina diploid nucleus. This “counting” mechanism has been proposed toinvolve a titration of the X-linked Jpx lncRNA and autosomally encodedCTCF protein (Sun et al., 2013). In parallel, a transient interaction(“pairing”) between two female X-chromosomes mediates the mutuallyexclusive choice of Xi and Xa (active X) (Bacher et al., 2006; Xu etal., 2006), with the subsequent action of Tsix lncRNA blocking XCI onthe designated Xa (Lee et al., 1999) and the action of Xist lncRNAinducing whole-chromosome silencing on the designated Xi (Brown et al.,1992; Penny et al., 1996). Xist spreads along the Xi and recruitssilencing complexes (Zhao et al., 2008; Wutz, 2011). With the exceptionof a small class of genes that escape XCI, nearly all 1000 genes on theXi are subject to silencing. Although significant progress has beenmade, many aspects of XCI mechanism continue to elude understanding.

TERRA's affinity for sex chromosomes led the present inventors tohypothesize that TERRA might have non-telomeric functions; based on itsassociation with sex chromosomes, possible roles surrounding the processof X-chromosome inactivation (XCI) were investigated. TERRA'sassociation with the X-chromosome provides a new and potentiallyrelevant avenue for exploration. Here we generate a map of TERRA'sgenomic binding sites, identify multiple non-telomeric targets, andinterrogate the relationship of X-linked target sites to sex chromosomebiology.

Here we have shown that TERRA function is not confined to telomeres, noris it cis-limited in action. TERRA is predominantly expressed from thesex chromosomes and originates at least in part from the sub-telomericregion known as the pseudoautosomal region (PAR). Several lines ofevidence argue that X- and P-linked “PAR-TERRA” is a continuoustranscript and that PAR-TERRA comprises a major subclass of telomericRNAs. First, similar results are obtained by Northern blot analysisusing PAR and TERRA oligo probes (FIG. 1F,G). Second, primer extensionindicates that TERRA and PAR RNA sequences are physically continuous(FIG. 1F). Third, RNA FISH experiments using TERRA and PAR probesdemonstrate overlapping RNA signals (FIG. 1A,C,E,H). Fourth, ChIRTanalysis indicates that nearly all TERRA-binding sites are alsoPAR-binding sites (FIG. 3,S3). Furthermore, knocking down PAR sequencesusing LNA gapmers results in TERRA depletion as well (FIG. 4A,B). FISHanalysis demonstrates that PAR-TERRA establishes a compartment next tothe telomeric ends of each sex chromosome, with a large PAR-TERRA RNAfocus localizing next to but not overlapping the Xist RNA domain.

We postulate that PAR-TERRA is as an organizing center for twoXCI-related processes (FIG. 6H-I, 7I). First, PAR-TERRA regulates generegulation on a global scale (FIG. 4). Altogether, we identifiedhundreds of TERRA-binding sites throughout the genome in MEFs, of which30-94 are X-linked. High PAR-TERRA coverage occurs near escapee genes,including genes of the pseudoautosomal region (FIG. 5). Perturbationexperiments demonstrate that X-linked PAR-TERRA sites promote expressionof escapee genes on the Xi. In the pseudoautosomal region, PAR-TERRAappears to protect genes from telomeric position effects. Analysis ofXist RNA localization indicated that Xist RNA is often enriched withindefined peaks near escapees (Simon et al., 2013), such as the prominentpeaks seen at Mid1 (FIG. 6A). These peaks suggest that Xist RNA may besequestered at “boundaries” near escapee genes and be prevented fromentering privileged loci. The idea of a boundary near escapees has beenexplored previously, with CTCF emerging as a candidate regulator(Filippova et al., 2005; Horvath et al., 2013). The loss of theseXist-enriched boundaries following PAR-TERRA depletion argues thatPAR-TERRA also aids in formation of the Xist boundaries. We thereforepropose a model in which PAR-TERRA holds Xist RNA in check and bringsescapee genes into a privileged juxta-telomeric compartment that ispermissive of transcription (FIG. 6H-I).

Methods of Reducing Expression of X-linked Escapee Genes

The present methods include using antisense oligonucleotides (ASO)against PAR-TERRA RNA to downregulate expression of escapee genes. Inhumans, all chromosomes may have the capacity to produce TERRAtranscripts, each from their own subtelomeric regions. Thesesubtelomeric regions are chromosome-specific; therefore, the X and Ysubtelomeric region (also called pseudoautosomal region) are distinctfrom autosomes. Sex-chromosome-specific effects can be achieved bytargeting the PAR end of the telomeric transcript. Without wishing to bebound by theory, knocking down PAR, TERRA, or PAR-TERRA with aninhibitory nucleic acids, e.g., an ASO, may disrupt the organizingcenter and thereby induce escapee gene downregulation. These inhibitorynucleic acids can therefore be used to treat disorders of sex chromosomeaneuploidy, e.g., Klinefelter Syndrome (XXY) and Triple X Syndrome(XXX), or any other condition that results in extra copies of all orpart of the X-chromosome (e.g., unbalanced X-autosome translocations).While individuals with extra X chromosomes are mostly dosage compensateddue to the counting mechanism (XXX women have two Xi's; XXY men have 1Xi), they have uncompensated dosage of the 15% of X-linked genes thatescape XCI.

Escapee genes include those listed in Table A.

TABLE E Escapee Genes Human Escapee Genes PR48; CALB3; SYAP1; HDHD1A;T54860; BC014382; AA348446; DKFZP564I1922; PRKX; Hs.431292; ITM2A;SRPX2; KIAA1817; MDS031; FLJ23018; HSU24186; Hs.271686; WBP5; TRPC5;TNFSF5; Hs.122516; Hs.404298; ARMCX4; FLJ11016; Hs.333016; DOCK11;LOC203427; CITED1; PLP1; PLCXD1; SLC25A6; LOC375793; ASMTL; DHRSX;FLJ43159; FLJ39679; CD99; XG; GYG2; ARSD; ARSE; Hs.399941; FLJ43700;AA971220; NLGN4X; FLJ12417; STS; Hs.186498; Hs.495638; PNPLA4;Hs.495641; Hs.348675; MGC17403; RAB9A; SEDL; Hs.41434; AA952971;FAM51A1; PIR; TMEM27; CA5BL; CA5B; AP1S2; Hs.121592; Hs.431654; CTPS2;Hs.431102; CXORF15; RBBP7; EIF1AX; EIF2S3; ZFX; Hs.458197; Hs.128084;USP9X; Hs.282780; Hs.86849; Hs.229338; DDX3X; MAOA; DUSP21; Hs.232417;AA130835; UBE1; INE1; JARID1C; A009X24; KIAA0522; Hs.87752; RPS4X; XIST;FLJ31610; F03810; PLXNB3; AVPR2; IKBKG; N74477; GPM6B; MGC39350; FUNDC1;SH3BGRL; L1CAM; GAB3; Hs.86443; TBL1X; GPR143; SMC1L1; RIBC1; Hs.258828;FLJ38564; NAP1L3; ZD89B07; SYTL4; Hs.527551~; ARHGAP4; RENBP; PCTK1;GRPR; CHM; HEIL2; HCFC1; OFD1; CRSP2; CLCN4; Hs.157695; MORF4L2; MYCL2;BRS3; ARD1; CXORF12; AF069137; Hs.108029; SH3KBP1; USP11; WAS; XEDAR;MAGEE1; ATP7A; Hs.445729; NXF3; LOC340544; PLS3; CUL4B; DXYS155E; MKRN4;23809; MSL3L1; ASB11; NHS; PHEX; TIMP1; MLLT7; PIN4; COX7B; RAB40A;COL4A6; FLJ36576; UTP14A; COVA1; PLAC1; LOC159090; MAGEA8; ABCD1; C6.1A;CLIC2; PDZK10; REPS2; CDKL5; Hs.435570; Hs.446513; RS1; PHKA2; N53651;Hs.444490; ACATE2; TAB3; Hs.177986; BCoR; SYP; CCNB3; LOC51248;FLJ20105; Hs.37464; ABCB7; Hs.182171; BTK; RPL36A; GLA; BEX1; FLJ21174;NXT2; FLJ22679; Hs.425072; AMMECR1; Hs.61094; PAK3; LHFPL1; FLJ22965;UPF3B; MCTS1; GPC4; PHF6; MOSPD1; Hs.436787; CDR1; SLITRK2; LOC347512~;ZNF185; M78874; HCA127; FLJ34366; FLJ12525; FMR1; IRAK1; TKTL1; VBP1;Hs.522189~; KIAA1280; MID1; Hs.187608; ARHGAP6; H48827; M62076; GLRA2;EUROIMAGE 35971; CXorf23; SAT; AA601738; DMD; AA461044; FLJ42925;TM4SF2; FLJ43479; ATP6AP2; ZC35F11; SLC9A7; RGN; SLC38A5; GATA1; KCND1;GRIPAP1; FLJ21687; HADH2; UREB1; DT1P1A10; FGD1; Hs.13041; LOC90736;MAGEH1; W68846; DKFZp686L07201; LOC92249; Hs.38448; MSN; STARD8; EFNB1;PJA1; ACRC; GPR23; FLJ13042; TM4SF6; ARMCX1; ARMCX2; Hs.53997; MGC23947;RAB9B; FLJ33516; CLDN2; AI650369; H66935; PRPS1; PSMD10; APG4A; CHRDL1;KLHL13; NKAP; PEPP-2~; ODZ1; XPNPEP2; CXorf9; Hs.269127; FHL1;Hs.205436; FLJ38034; ATP11C; Hs.112784; Hs.127679; Hs.31542; LDOC1;CD99L2; PNMA5; SYBL1; G06389; TIMM8A; HPRT1; FAM9C; AW448933; UBQLN2;FLJ31204; PGPL; SHOX; ZBED1; KAL1; IL9R; IL9R Mouse Escapee Genes1810030O07Rik; 5530601H04Rik; 5730416F02Rik; Abcb7; Aff2; Bgn; Car5b;Col4a6; Cox7b; Cxx1b; Ddx3x; Dkc1; Dmd; Dusp9; Eda; Eif2s3x; Fgf13;Firre; Frmpd4; Ftx; Gm5124; Gyk; Hmgb3; Il1rapl1; Irak1; Jpx; Kdm5c;Kdm6a; Kif4; Lage3; Mageb16; Mageb18; Mbtps2; Mid1; Msn; Naa10; Ndufb11;Ngfrap1; Nono; Pbdc1; Pdha1; Plp2; Pola1; Praf2; Prickle3; Rbbp7; Rbm10;Reps2; Rpl39; Rps4x; Sept6; Shroom4; Slc16a2; Slc25a5; Usp11; Wbp5;Xist; Flna; Ikbkg; Hcfc1; Huwe1; Maged1; Ogt; Asmt; and Erdr1 # 305escapees in human, and 65 escapees in mouse # human escapees defined asXi/Xa > 0.1 based on allele specific PCR in this paper: (Nature, 2005,Laura Carrel1 & Huntington F. Willard) X-inactivation profile revealsextensive variability in X-linked gene expression in females #mouseescapees defined as Xi/Xa > 0.1 or Xi read counts >5 in MEF RNA-seq

Table B provides a list of escapee genes in the human pseudoautosomalregion (PAR) of the X chromosome. Annotated gene name and full genedescription are shown. Whether or not there is a Y-chromosome homologueis noted in the third column.

TABLE B Human PAR genes or Y orthologues or pseuodogene PR48 Proteinphosphatase 2A 48 kDa subunit Pseudoautosomal; Y identity PLCXD1Phosphatidylinositol phospholipase C, X domain 1 Pseudoautosomal; Yidentity SLC25A6 Solute carrier family 25, member 6 Pseudoautosomal; Yidentity LOC375793 Hypothetical protein with EST supportPseudoautosomal; Y identity ASMTL AcetylserotoninO-methyltransferase-like Pseudoautosomal; Y identity DHRSXDehydrogenase/reductase (SDR) family Pseudoautosomal; Y identityFLJ43159 mRNA of unknown function Pseudoautosomal; Y identity FLJ39679mRNA of unknown function Pseudoautosomal; Y identity CD99 CD99 antigenPseudoautosomal; Y identity DXYS155E Lymphocyte surface proteinPseudoautosomal; Y identity PGPL Pseudoautosomal GTP-bindingprotein-like Pseudoautosomal; Y identity SHOX Short stature homeoboxPseudoautosomal; Y identity ZBED1 zinc finger, BED domain containing 1Pseudoautosomal; Y identity SYBL1 Synaptobrevin-like 1 Pseudoautosomal;Y identity IL9R Interleukin 9 receptor Pseudoautosomal; Y identity RPS4XRibosomal protein S4, X isoform Y orthologue UBE1 Ubiquitin-activatingenzyme E1 Ancestral Y homolog JARID1C Jumonji, AT rich interactivedomain 1C Y orthologue RAB9A Ras-related GTP-binding protein Y homologyby BLAST SEDL Spondyloepiphyseal dysplasia, late Y pseudogene CXORF15Chromosome X open reading frame 15 Y orthologues EIF1AX Eukaryotictranslation initiation factor 4C Y orthologue ZFX Zinc finger proteinX-linked Y orthologue USP9X Ubiquitin specific protease 9, X isoform Yorthologue DDX3X DEAD/H box 3, X-linked Y orthologue DUSP21 Dualspecificity phosphatase 21 Y orthologue OFD1 Oral-facial-digitalsyndrome 1 gene Y pseudogene CRSP2 Cofactor required for Sp1transcriptional activation Y pseudogene TAB3 TAK1-binding protein 3 Ypseudogene BCoR BCL6 co-repressor Y pseudogene FAM9C Family withsequence similarity 9, member C Y homology by BLAST HDHD1A Haloaciddehalogenase-like hydrolase domain 1 Y pseudogene NLGN4X Neuroligin 4 Yorthologue STS Steroid sulfatase Y pseudogene TBL1X Transducin (beta)like 1 Y orthologue GPR143 G protein-coupled receptor 143 Y pseudogeneKAL1 Kallmann syndrome 1 sequence Y pseudogene AA348446 ESTs Y homologyby BLAST DKFZP564I1922 Adlican Y pseudogene PRKX Protein kinase,X-linked Y orthologue XG Xg blood group Y pseudogene GYG2 Glycogenin 2 Ypseudogene ARSD Arylsulfatase D Y pseudogene ARSE Arylsulfatase E Ypseudogene Hs.399941 ESTs Y homology by BLAST FLJ43700 Hypotheticalprotein with mRNA & EST support Y homology by BLAST

The sequence of human PAR-TERRA is provided herewith as SEQ ID NO:1. Thesequence of mouse PAR-TERRA is provided herewith as SED ID NO:2. In someembodiments, the sequence of an oligo targeting TERRA is 5′-TAA CCC TAACCC TAA C-3′ (SEQ ID NO:5); or PAR-TERRA is 5′-TCT CTG TCT CTG TCG C-3′(SEQ ID NO:6).

Method of Reducing Expression of from non Xi Genes

TERRA forms a special compartment for gene activation, not only forescapees on the Xi but also for autosomal genes and subtelomeric geneson autosomal ends, of which Chr 1, 3, 4, 8, 18, and 19 are shown herein(see, e.g., FIGS. 4, 5A-B). PAR-TERRA and various Chromosome-specificTERRAs (produced from the subtelomeric regions) are also used toregulate genes outside of the Xi. The present methods can be applied todownregulate a network of Xa and subtelomeric autosomal genes involvedin growth control and apoptosis, and other processes relevant to humandisease. Two examples include active X (Xa) genes and the FSHDsubtelomeric region of human Chromosome 4. The Xa also producesPAR-TERRA from its pseudoautosomal region and has multiple targetsoutside of its pseudoautosomal region. Targeting PAR, TERRA, orPAR-TERRA can reduce expression from the Xa. PAR-TERRA and TERRA alsotarget thousands of autosomal sites with closely linked genes. Aspecific example include the Chr4 region associated withfacioscapulohumeral muscular dystrophy (FSHD), which is located in thesubtelomeric region of human Chr4 and contains coding genes FRG1, FRG2,DUX4, and the long noncoding RNAs of forward and reverse orientationsfrom the macrosatellite repeat, D4Z4. FSHD is caused by ectopicexpression of these genes when the D4Z4 repeat contracts and becomes“activated”. Thus, PAR, TERRA, or PAR-TERRA or Chr4-specific TERRA couldbe targeted to downregulated the associated subtelomeric genes.

In addition, PAR-TERRA knockdown resulted in downregulation of genesenriched for cell cycle and apoptosis genes (see Example 5). Thus,targeting PAR, TERRA, or PAR-TERRA transcripts can be an effectivemethod of treating cancer and other human diseases where theX-chromosome and various growth control genes are frequentlyoverexpressed. The sequence of human Chr4 FSHD region is providedherewith as SEQ ID NO:3.

Disorders of Sex Chromosome Aneuploidy

The present methods can be used to reduce expression of escapee genes insubjects with disorders of sex chromosome aneuploidy in which at leastone extra X chromosome is present (referred to herein as a supernumeraryX chromosome). The term Klinefelter syndrome (KS) describes a group ofdisorders in which at least one extra X chromosome is present inaddition to a normal male karyotype, referred to in standard geneticsnomenclature as 46,XY. Related to the KS group is 47,XXY aneuploidy,which is the most prevalent disorder of sex chromosomes in humans with aprevalence of about 1:500. Rarer sex chromosome aneuploidies include48,XXYY and 48,XXXY (about 1:17,000 to 1:50,000); 49,XXXXY (about1:85,000 to 1:100,000) births. See, e.g., Visootsak and Graham, OrphanetJ Rare Dis. 2006; 1: 42; Targaltia et al., Acta Paediatr. 2011 June;100(6):851-60). Triple X syndrome (47,XXX) is a disorder in which atleast one extra X chromosome is present in addition to a normal femalekaryotype; 48,XXXX and 49,XXXXX have also been described (Schoubben etal., Eur J Pediatr. 2011 October; 170(10):1325-7). Conditions resultingfrom unbalanced X-autosome translocations or cancers (and other humandiseases) with X-chromosomal aneuploidies may be treated similarly usingthe technology. These conditions can result in a large number ofdeleterious physical, psychological, and intellectual effects inaffected individuals (see, e.g., Visootsak and Graham, Orphanet J RareDis. 2006; 1: 42; Schoubben et al., Eur J Pediatr. 2011 October;170(10):1325-7; Targaltia et al., Acta Paediatr. 2011 June;100(6):851-60).

Inhibitory Nucleic Acids Targeting PAR-TERRA or OtherChromosome-Specific TERRA

The methods and compositions described herein can include nucleic acidssuch as a small inhibitory RNA (siRNA) or LNA that targets (specificallybinds, or is complementary to) PAR, PAR-TERRA, or otherchromosome-specific TERRA (e.g., Chr4-specific, as produced from thesubtelomeric region of human Chr4 which is associated withfacioscapulohumeral muscular dystrophy (FSHD)) RNA. Inhibitory nucleicacids useful in the present methods and compositions include antisenseoligonucleotides, ribozymes, external guide sequence (EGS)oligonucleotides, siRNA compounds, single- or double-stranded RNAinterference (RNAi) compounds such as siRNA compounds, moleculescomprising modified bases, locked nucleic acid molecules (LNAmolecules), antagomirs, peptide nucleic acid molecules (PNA molecules),and other oligomeric compounds or oligonucleotide mimetics whichhybridize to at least a portion of the target nucleic acid and modulateits function. In some embodiments, the inhibitory nucleic acids includeantisense RNA, antisense DNA, chimeric antisense oligonucleotides,antisense oligonucleotides comprising modified linkages, interferenceRNA (RNAi), short interfering RNA (siRNA); a micro, interfering RNA(miRNA); a small, temporal RNA (stRNA); or a short, hairpin RNA (shRNA);small RNA-induced gene activation (RNAa); small activating RNAs(saRNAs), or combinations thereof. See, e.g., U.S. Ser. No. 62/010,342,WO 2012/065143, WO 2012/087983, and WO 2014/025887. However, in someembodiments the inhibitory nucleic acid is not an miRNA, an stRNA, anshRNA, an siRNA, an RNAi, or a dsRNA.

In some embodiments, the inhibitory nucleic acids are 10 to 50, 10 to20, 10 to 25, 13 to 50, or 13 to 30 nucleotides in length. One havingordinary skill in the art will appreciate that this embodies inhibitorynucleic acids having complementary portions of 10, 11, 12, 13, 14, 15,16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33,34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50nucleotides in length, or any range therewithin. In some embodiments,the inhibitory nucleic acids are 15 nucleotides in length. In someembodiments, the inhibitory nucleic acids are 12 or 13 to 20, 25, or 30nucleotides in length. One having ordinary skill in the art willappreciate that this embodies inhibitory nucleic acids havingcomplementary portions of 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22,23, 24, 25, 26, 27, 28, 29 or 30 nucleotides in length, or any rangetherewithin (complementary portions refers to those portions of theinhibitory nucleic acids that are complementary to the target sequence).

The inhibitory nucleic acids useful in the present methods aresufficiently complementary to the target RNA, i.e., hybridizesufficiently well and with sufficient specificity, to give the desiredeffect. “Complementary” refers to the capacity for pairing, throughhydrogen bonding, between two sequences comprising naturally ornon-naturally occurring bases or analogs thereof. For example, if a baseat one position of an inhibitory nucleic acid is capable of hydrogenbonding with a base at the corresponding position of a RNA, then thebases are considered to be complementary to each other at that position.100% complementarity is not required.

Routine methods can be used to design an inhibitory nucleic acid thatbinds to the target sequence with sufficient specificity. In someembodiments, the methods include using bioinformatics methods known inthe art to identify regions of secondary structure, e.g., one, two, ormore stem-loop structures, or pseudoknots, and selecting those regionsto target with an inhibitory nucleic acid. For example, “gene walk”methods can be used to optimize the inhibitory activity of the nucleicacid; for example, a series of oligonucleotides of 10-30 nucleotidesspanning the length of a target RNA can be prepared, followed by testingfor activity. Optionally, gaps, e.g., of 5-10 nucleotides or more, canbe left between the target sequences to reduce the number ofoligonucleotides synthesized and tested. GC content is preferablybetween about 30-60%. Contiguous runs of three or more Gs or Cs shouldbe avoided where possible (for example, it may not be possible with veryshort (e.g., about 9-10 nt) oligonucleotides).

In some embodiments, the inhibitory nucleic acid molecules can bedesigned to target a specific region of the RNA sequence. For example, aspecific functional region can be targeted, e.g., a region comprising aknown RNA localization motif (i.e., a region complementary to the targetnucleic acid on which the RNA acts). Alternatively or in addition,highly conserved regions can be targeted, e.g., regions identified byaligning sequences from disparate species such as primate (e.g., human)and rodent (e.g., mouse) and looking for regions with high degrees ofidentity. Percent identity can be determined routinely using basic localalignment search tools (BLAST programs) (Altschul et al., J. Mol. Biol.,1990, 215, 403-410; Zhang and Madden, Genome Res., 1997, 7, 649-656),e.g., using the default parameters.

Once one or more target regions, segments or sites have been identified,e.g., within a sequence known in the art or provided herein, inhibitorynucleic acid compounds are chosen that are sufficiently complementary tothe target, i.e., that hybridize sufficiently well and with sufficientspecificity (i.e., do not substantially bind to other non-target RNAs),to give the desired effect.

In the context of this invention, hybridization means hydrogen bonding,which may be Watson-Crick, Hoogsteen or reversed Hoogsteen hydrogenbonding, between complementary nucleoside or nucleotide bases. Forexample, adenine and thymine are complementary nucleobases which pairthrough the formation of hydrogen bonds. Complementary, as used herein,refers to the capacity for precise pairing between two nucleotides. Forexample, if a nucleotide at a certain position of an oligonucleotide iscapable of hydrogen bonding with a nucleotide at the same position of aRNA molecule, then the inhibitory nucleic acid and the RNA areconsidered to be complementary to each other at that position. Theinhibitory nucleic acids and the RNA are complementary to each otherwhen a sufficient number of corresponding positions in each molecule areoccupied by nucleotides which can hydrogen bond with each other. Thus,“specifically hybridisable” and “complementary” are terms which are usedto indicate a sufficient degree of complementarity or precise pairingsuch that stable and specific binding occurs between the inhibitorynucleic acid and the RNA target. For example, if a base at one positionof an inhibitory nucleic acid is capable of hydrogen bonding with a baseat the corresponding position of a RNA, then the bases are considered tobe complementary to each other at that position. 100% complementarity isnot required.

It is understood in the art that a complementary nucleic acid sequenceneed not be 100% complementary to that of its target nucleic acid to bespecifically hybridisable. A complementary nucleic acid sequence forpurposes of the present methods is specifically hybridisable whenbinding of the sequence to the target RNA molecule interferes with thenormal function of the target RNA to cause a loss of activity, and thereis a sufficient degree of complementarity to avoid non-specific bindingof the sequence to non-target RNA sequences under conditions in whichspecific binding is desired, e.g., under physiological conditions in thecase of in vivo assays or therapeutic treatment, and in the case of invitro assays, under conditions in which the assays are performed undersuitable conditions of stringency. For example, stringent saltconcentration will ordinarily be less than about 750 mM NaCl and 75 mMtrisodium citrate, preferably less than about 500 mM NaCl and 50 mMtrisodium citrate, and more preferably less than about 250 mM NaCl and25 mM trisodium citrate. Low stringency hybridization can be obtained inthe absence of organic solvent, e.g., formamide, while high stringencyhybridization can be obtained in the presence of at least about 35%formamide, and more preferably at least about 50% formamide. Stringenttemperature conditions will ordinarily include temperatures of at leastabout 30° C., more preferably of at least about 37° C., and mostpreferably of at least about 42° C. Varying additional parameters, suchas hybridization time, the concentration of detergent, e.g., sodiumdodecyl sulfate (SDS), and the inclusion or exclusion of carrier DNA,are well known to those skilled in the art. Various levels of stringencyare accomplished by combining these various conditions as needed. In apreferred embodiment, hybridization will occur at 30° C. in 750 mM NaCl,75 mM trisodium citrate, and 1% SDS. In a more preferred embodiment,hybridization will occur at 37° C. in 500 mM NaCl, 50 mM trisodiumcitrate, 1% SDS, 35% formamide, and 100 μg/ml denatured salmon sperm DNA(ssDNA). In a most preferred embodiment, hybridization will occur at 42°C. in 250 mM NaCl, 25 mM trisodium citrate, 1% SDS, 50% formamide, and200 μg/ml ssDNA. Useful variations on these conditions will be readilyapparent to those skilled in the art.

For most applications, washing steps that follow hybridization will alsovary in stringency. Wash stringency conditions can be defined by saltconcentration and by temperature. As above, wash stringency can beincreased by decreasing salt concentration or by increasing temperature.For example, stringent salt concentration for the wash steps willpreferably be less than about 30 mM NaCl and 3 mM trisodium citrate, andmost preferably less than about 15 mM NaCl and 1.5 mM trisodium citrate.Stringent temperature conditions for the wash steps will ordinarilyinclude a temperature of at least about 25° C., more preferably of atleast about 42° C., and even more preferably of at least about 68° C. Ina preferred embodiment, wash steps will occur at 25° C. in 30 mM NaCl, 3mM trisodium citrate, and 0.1% SDS. In a more preferred embodiment, washsteps will occur at 42° C. in 15 mM NaCl, 1.5 mM trisodium citrate, and0.1% SDS. In a more preferred embodiment, wash steps will occur at 68°C. in 15 mM NaCl, 1.5 mM trisodium citrate, and 0.1% SDS. Additionalvariations on these conditions will be readily apparent to those skilledin the art. Hybridization techniques are well known to those skilled inthe art and are described, for example, in Benton and Davis (Science196:180, 1977); Grunstein and Hogness (Proc. Natl. Acad. Sci., USA72:3961, 1975); Ausubel et al. (Current Protocols in Molecular Biology,Wiley Interscience, New York, 2001); Berger and Kimmel (Guide toMolecular Cloning Techniques, 1987, Academic Press, New York); andSambrook et al., Molecular Cloning: A Laboratory Manual, Cold SpringHarbor Laboratory Press, New York.

In general, the inhibitory nucleic acids useful in the methods describedherein have at least 80% sequence complementarity to a target regionwithin the target nucleic acid, e.g., 90%, 95%, or 100% sequencecomplementarity to the target region within an RNA. For example, anantisense compound in which 18 of 20 nucleobases of the antisenseoligonucleotide are complementary, and would therefore specificallyhybridize, to a target region would represent 90 percentcomplementarity. Percent complementarity of an inhibitory nucleic acidwith a region of a target nucleic acid can be determined routinely usingbasic local alignment search tools (BLAST programs) (Altschul et al., J.Mol. Biol., 1990, 215, 403-410; Zhang and Madden, Genome Res., 1997, 7,649-656). Inhibitory nucleic acids that hybridize to an RNA can beidentified through routine experimentation. In general the inhibitorynucleic acids must retain specificity for their target, i.e., must notdirectly bind to, or directly significantly affect expression levels of,transcripts other than the intended target.

For further disclosure regarding inhibitory nucleic acids, please seeUS2010/0317718 (antisense oligos); US2010/0249052 (double-strandedribonucleic acid (dsRNA)); US2009/0181914 and US2010/0234451 (LNAs);US2007/0191294 (siRNA analogues); US2008/0249039 (modified siRNA); andWO2010/129746 and WO2010/040112 (inhibitory nucleic acids), as well asWO 2012/065143, WO 2012/087983, and WO 2014/025887 (inhibitory nucleicacids targeting non-coding RNAs/supRNAss), all of which are incorporatedherein by reference in their entirety.

Antisense

In some embodiments, the inhibitory nucleic acids are antisenseoligonucleotides. Antisense oligonucleotides are typically designed toblock expression of a DNA or RNA target by binding to the target and(without wishing to be bound by theory) halting expression at the levelof transcription, translation, or splicing. Antisense oligonucleotidesof the present invention are complementary nucleic acid sequencesdesigned to hybridize under stringent conditions to an RNA. Thus,oligonucleotides are chosen that are sufficiently complementary to thetarget, i.e., that hybridize sufficiently well and with sufficientspecificity, to give the desired effect.

siRNA/shRNA

In some embodiments, the nucleic acid sequence that is complementary toan target RNA can be an interfering RNA, including but not limited to asmall interfering RNA (“siRNA”) or a small hairpin RNA (“shRNA”).Methods for constructing interfering RNAs are well known in the art. Forexample, the interfering RNA can be assembled from two separateoligonucleotides, where one strand is the sense strand and the other isthe antisense strand, wherein the antisense and sense strands areself-complementary (i.e., each strand comprises nucleotide sequence thatis complementary to nucleotide sequence in the other strand; such aswhere the antisense strand and sense strand form a duplex or doublestranded structure); the antisense strand comprises nucleotide sequencethat is complementary to a nucleotide sequence in a target nucleic acidmolecule or a portion thereof (i.e., an undesired gene) and the sensestrand comprises nucleotide sequence corresponding to the target nucleicacid sequence or a portion thereof. Alternatively, interfering RNA isassembled from a single oligonucleotide, where the self-complementarysense and antisense regions are linked by means of nucleic acid based ornon-nucleic acid-based linker(s). The interfering RNA can be apolynucleotide with a duplex, asymmetric duplex, hairpin or asymmetrichairpin secondary structure, having self-complementary sense andantisense regions, wherein the antisense region comprises a nucleotidesequence that is complementary to nucleotide sequence in a separatetarget nucleic acid molecule or a portion thereof and the sense regionhaving nucleotide sequence corresponding to the target nucleic acidsequence or a portion thereof. The interfering can be a circularsingle-stranded polynucleotide having two or more loop structures and astem comprising self-complementary sense and antisense regions, whereinthe antisense region comprises nucleotide sequence that is complementaryto nucleotide sequence in a target nucleic acid molecule or a portionthereof and the sense region having nucleotide sequence corresponding tothe target nucleic acid sequence or a portion thereof, and wherein thecircular polynucleotide can be processed either in vivo or in vitro togenerate an active siRNA molecule capable of mediating RNA interference.

In some embodiments, the interfering RNA coding region encodes aself-complementary RNA molecule having a sense region, an antisenseregion and a loop region. Such an RNA molecule when expressed desirablyforms a “hairpin” structure, and is referred to herein as an “shRNA.”The loop region is generally between about 2 and about 10 nucleotides inlength. In some embodiments, the loop region is from about 6 to about 9nucleotides in length. In some embodiments, the sense region and theantisense region are between about 15 and about 20 nucleotides inlength. Following post-transcriptional processing, the small hairpin RNAis converted into a siRNA by a cleavage event mediated by the enzymeDicer, which is a member of the RNase III family. The siRNA is thencapable of inhibiting the expression of a gene with which it shareshomology. For details, see Brummelkamp et al., Science 296:550-553,(2002); Lee et al, Nature Biotechnol., 20, 500-505, (2002); Miyagishiand Taira, Nature Biotechnol 20:497-500, (2002); Paddison et al. Genes &Dev. 16:948-958, (2002); Paul, Nature Biotechnol, 20, 505-508, (2002);Sui, Proc. Natl. Acad. Sd. USA, 99(6), 5515-5520, (2002); Yu et al. ProcNatlAcadSci USA 99:6047-6052, (2002).

The target RNA cleavage reaction guided by siRNAs is highly sequencespecific. In general, siRNA containing a nucleotide sequences identicalto a portion of the target nucleic acid are preferred for inhibition.However, 100% sequence identity between the siRNA and the target gene isnot required to practice the present invention. Thus the invention hasthe advantage of being able to tolerate sequence variations that mightbe expected due to genetic mutation, strain polymorphism, orevolutionary divergence. For example, siRNA sequences with insertions,deletions, and single point mutations relative to the target sequencehave also been found to be effective for inhibition. Alternatively,siRNA sequences with nucleotide analog substitutions or insertions canbe effective for inhibition. In general the siRNAs must retainspecificity for their target, i.e., must not directly bind to, ordirectly significantly affect expression levels of, transcripts otherthan the intended target.

Ribozymes

Trans-cleaving enzymatic nucleic acid molecules can also be used; theyhave shown promise as therapeutic agents for human disease (Usman &McSwiggen, 1995 Ann. Rep. Med. Chem. 30, 285-294; Christoffersen andMarr, 1995 J. Med. Chem. 38, 2023-2037). Enzymatic nucleic acidmolecules can be designed to cleave specific RNA targets within thebackground of cellular RNA. Such a cleavage event renders the RNAnon-functional.

In general, enzymatic nucleic acids with RNA cleaving activity act byfirst binding to a target RNA. Such binding occurs through the targetbinding portion of a enzymatic nucleic acid which is held in closeproximity to an enzymatic portion of the molecule that acts to cleavethe target RNA. Thus, the enzymatic nucleic acid first recognizes andthen binds a target RNA through complementary base pairing, and oncebound to the correct site, acts enzymatically to cut the target RNA.Strategic cleavage of such a target RNA will destroy its ability todirect synthesis of an encoded protein. After an enzymatic nucleic acidhas bound and cleaved its RNA target, it is released from that RNA tosearch for another target and can repeatedly bind and cleave newtargets.

Several approaches such as in vitro selection (evolution) strategies(Orgel, 1979, Proc. R. Soc. London, B 205, 435) have been used to evolvenew nucleic acid catalysts capable of catalyzing a variety of reactions,such as cleavage and ligation of phosphodiester linkages and amidelinkages, (Joyce, 1989, Gene, 82, 83-87; Beaudry et al., 1992, Science257, 635-641; Joyce, 1992, Scientific American 267, 90-97; Breaker etal, 1994, TIBTECH 12, 268; Bartel et al, 1993, Science 261:1411-1418;Szostak, 1993, TIBS 17, 89-93; Kumar et al, 1995, FASEB J., 9, 1183;Breaker, 1996, Curr. Op. Biotech., 1, 442). The development of ribozymesthat are optimal for catalytic activity would contribute significantlyto any strategy that employs RNA-cleaving ribozymes for the purpose ofregulating gene expression. The hammerhead ribozyme, for example,functions with a catalytic rate (kcat) of about 1 min⁻¹ in the presenceof saturating (10 mM) concentrations of Mg²⁺ cofactor. An artificial“RNA ligase” ribozyme has been shown to catalyze the correspondingself-modification reaction with a rate of about 100 min⁻¹. In addition,it is known that certain modified hammerhead ribozymes that havesubstrate binding arms made of DNA catalyze RNA cleavage with multipleturn-over rates that approach 100 min⁻¹.

Modified Inhibitory Nucleic Acids

In some embodiments, the inhibitory nucleic acids used in the methodsdescribed herein are modified, e.g., comprise one or more modified bondsor bases. A number of modified bases include phosphorothioate,methylphosphonate, peptide nucleic acids, or locked nucleic acid (LNA)molecules. Some inhibitory nucleic acids are fully modified, whileothers are chimeric and contain two or more chemically distinct regions,each made up of at least one nucleotide. These inhibitory nucleic acidstypically contain at least one region of modified nucleotides thatconfers one or more beneficial properties (such as, for example,increased nuclease resistance, increased uptake into cells, increasedbinding affinity for the target) and a region that is a substrate forenzymes capable of cleaving RNA:DNA or RNA:RNA hybrids. Chimericinhibitory nucleic acids of the invention may be formed as compositestructures of two or more oligonucleotides, modified oligonucleotides,oligonucleosides and/or oligonucleotide mimetics as described above.Such compounds have also been referred to in the art as hybrids orgapmers. Representative United States patents that teach the preparationof such hybrid structures comprise, but are not limited to, U.S. Pat.Nos. 5,013,830; 5,149,797; 5, 220,007; 5,256,775; 5,366,878; 5,403,711;5,491,133; 5,565,350; 5,623,065; 5,652,355; 5,652,356; and 5,700,922,each of which is herein incorporated by reference.

In some embodiments, the inhibitory nucleic acid comprises at least onenucleotide modified at the 2′ position of the sugar, most preferably a2′-O-alkyl, 2′-O-alkyl-O-alkyl or 2′-fluoro-modified nucleotide. Inother preferred embodiments, RNA modifications include 2′-fluoro,2′-amino and 2′ O-methyl modifications on the ribose of pyrimidines,abasic residues or an inverted base at the 3′ end of the RNA. Suchmodifications are routinely incorporated into oligonucleotides and theseoligonucleotides have been shown to have a higher Tm (i.e., highertarget binding affinity) than; 2′-deoxyoligonucleotides against a giventarget.

A number of nucleotide and nucleoside modifications have been shown tomake the inhibitory nucleic acid into which they are incorporated moreresistant to nuclease digestion than the native oligodeoxynucleotide;these modified oligos survive intact for a longer time than unmodifiedinhibitory nucleic acids. Specific examples of modified inhibitorynucleic acids include those comprising modified backbones, for example,phosphorothioates, phosphotriesters, methyl phosphonates, short chainalkyl or cycloalkyl intersugar linkages or short chain heteroatomic orheterocyclic intersugar linkages. Most preferred are inhibitory nucleicacids with phosphorothioate backbones and those with heteroatombackbones, particularly CH2—NH—O—CH2, CH, ˜N(CH3)˜O˜CH2 (known as amethylene(methylimino) or MMI backbone], CH2 —O—N (CH3)—CH2, CH2—N(CH3)—N (CH3)—CH2 and O—N (CH3)—CH2 —CH2 backbones, wherein the nativephosphodiester backbone is represented as O—P—O—CH,); amide backbones(see De Mesmaeker et al. Ace. Chem. Res. 1995, 28:366-374); morpholinobackbone structures (see Summerton and Weller, U.S. Pat. No. 5,034,506);peptide nucleic acid (PNA) backbone (wherein the phosphodiester backboneof the inhibitory nucleic acid is replaced with a polyamide backbone,the nucleotides being bound directly or indirectly to the aza nitrogenatoms of the polyamide backbone, see Nielsen et al., Science 1991, 254,1497). Phosphorus-containing linkages include, but are not limited to,phosphorothioates, chiral phosphorothioates, phosphorodithioates,phosphotriesters, aminoalkylphosphotriesters, methyl and other alkylphosphonates comprising 3′alkylene phosphonates and chiral phosphonates,phosphinates, phosphoramidates comprising 3′-amino phosphoramidate andaminoalkylphosphoramidates, thionophosphoramidates,thionoalkylphosphonates, thionoalkylphosphotriesters, andboranophosphates having normal 3′-5′ linkages, 2′-5′ linked analogs ofthese, and those having inverted polarity wherein the adjacent pairs ofnucleoside units are linked 3′-5′ to 5′-3′ or 2′-5′ to 5′-2′; see U.S.Pat. Nos. 3,687,808; 4,469,863; 4,476,301; 5,023,243; 5,177,196;5,188,897; 5,264,423; 5,276,019; 5,278,302; 5,286,717; 5,321,131;5,399,676; 5,405,939; 5,453,496; 5,455,233; 5,466,677; 5,476,925;5,519,126; 5,536,821; 5,541,306; 5,550,111; 5,563,253; 5,571,799;5,587,361; and 5,625,050.

Morpholino-based oligomeric compounds are described in Dwaine A. Braaschand David R. Corey, Biochemistry, 2002, 41(14), 4503-4510); Genesis,volume 30, issue 3, 2001; Heasman, J., Dev. Biol., 2002, 243, 209-214;Nasevicius et al., Nat. Genet., 2000, 26, 216-220; Lacerra et al., Proc.Natl. Acad. Sci., 2000, 97, 9591-9596; and U.S. Pat. No. 5,034,506,issued Jul. 23, 1991.

Cyclohexenyl nucleic acid inhibitory nucleic acid mimetics are describedin Wang et al., J. Am. Chem. Soc., 2000, 122, 8595-8602.

Modified inhibitory nucleic acid backbones that do not include aphosphorus atom therein have backbones that are formed by short chainalkyl or cycloalkyl internucleoside linkages, mixed heteroatom and alkylor cycloalkyl internucleoside linkages, or one or more short chainheteroatomic or heterocyclic internucleoside linkages. These comprisethose having morpholino linkages (formed in part from the sugar portionof a nucleoside); siloxane backbones; sulfide, sulfoxide and sulfonebackbones; formacetyl and thioformacetyl backbones; methylene formacetyland thioformacetyl backbones; alkene containing backbones; sulfamatebackbones; methyleneimino and methylenehydrazino backbones; sulfonateand sulfonamide backbones; amide backbones; and others having mixed N,O, S and CH2 component parts; see U.S. Pat. Nos. 5,034,506; 5,166,315;5,185,444; 5,214,134; 5,216,141; 5,235,033; 5,264,562; 5,264,564;5,405,938; 5,434,257; 5,466,677; 5,470,967; 5,489,677; 5,541,307;5,561,225; 5,596,086; 5,602,240; 5,610,289; 5,602,240; 5,608,046;5,610,289; 5,618,704; 5,623,070; 5,663,312; 5,633,360; 5,677,437; and5,677,439, each of which is herein incorporated by reference.

One or more substituted sugar moieties can also be included, e.g., oneof the following at the 2′ position: OH, SH, SCH₃, F, OCN, OCH₃ OCH₃,OCH₃ O(CH₂)n CH₃, O(CH₂)n NH₂ or O(CH₂)n CH₃ where n is from 1 to about10; Ci to C10 lower alkyl, alkoxyalkoxy, substituted lower alkyl,alkaryl or aralkyl; Cl; Br; CN; CF3; OCF3; O-, S-, or N-alkyl; O-, S-,or N-alkenyl; SOCH3; SO2 CH3; ONO2; NO2; N3; NH2; heterocycloalkyl;heterocycloalkaryl; aminoalkylamino; polyalkylamino; substituted silyl;an RNA cleaving group; a reporter group; an intercalator; a group forimproving the pharmacokinetic properties of an inhibitory nucleic acid;or a group for improving the pharmacodynamic properties of an inhibitorynucleic acid and other substituents having similar properties. Apreferred modification includes 2′-methoxyethoxy [2′-0-CH₂CH₂OCH₃, alsoknown as 2′-O-(2-methoxyethyl)] (Martin et al, HeIv. Chim. Acta, 1995,78, 486). Other preferred modifications include 2′-methoxy (2′-0-CH₃),2′-propoxy (2′-OCH₂ CH₂CH₃) and 2′-fluoro (2′-F). Similar modificationsmay also be made at other positions on the inhibitory nucleic acid,particularly the 3′ position of the sugar on the 3′ terminal nucleotideand the 5′ position of 5′ terminal nucleotide. Inhibitory nucleic acidsmay also have sugar mimetics such as cyclobutyls in place of thepentofuranosyl group.

Inhibitory nucleic acids can also include, additionally oralternatively, nucleobase (often referred to in the art simply as“base”) modifications or substitutions. As used herein, “unmodified” or“natural” nucleobases include adenine (A), guanine (G), thymine (T),cytosine (C) and uracil (U). Modified nucleobases include nucleobasesfound only infrequently or transiently in natural nucleic acids, e.g.,hypoxanthine, 6-methyladenine, 5-Me pyrimidines, particularly5-methylcytosine (also referred to as 5-methyl-2′ deoxycytosine andoften referred to in the art as 5-Me-C), 5-hydroxymethylcytosine (HMC),glycosyl HMC and gentobiosyl HMC, as well as synthetic nucleobases,e.g., 2-aminoadenine, 2-(methylamino)adenine,2-(imidazolylalkyl)adenine, 2-(aminoalklyamino)adenine or otherheterosubstituted alkyladenines, 2-thiouracil, 2-thiothymine,5-bromouracil, 5-hydroxymethyluracil, 8-azaguanine, 7-deazaguanine, N6(6-aminohexyl)adenine and 2,6-diaminopurine. Kornberg, A., DNAReplication, W. H. Freeman & Co., San Francisco, 1980, pp75-77;Gebeyehu, G., et al. Nucl. Acids Res. 1987, 15:4513). A “universal” baseknown in the art, e.g., inosine, can also be included. 5-Me-Csubstitutions have been shown to increase nucleic acid duplex stabilityby 0.6-1.2<0>C. (Sanghvi, Y. S., in Crooke, S. T. and Lebleu, B., eds.,Antisense Research and Applications, CRC Press, Boca Raton, 1993, pp.276-278) and are presently preferred base substitutions.

It is not necessary for all positions in a given inhibitory nucleic acidto be uniformly modified, and in fact more than one of theaforementioned modifications may be incorporated in a single inhibitorynucleic acid or even at within a single nucleoside within an inhibitorynucleic acid.

In some embodiments, both a sugar and an internucleoside linkage, i.e.,the backbone, of the nucleotide units are replaced with novel groups.The base units are maintained for hybridization with an appropriatenucleic acid target compound. One such oligomeric compound, aninhibitory nucleic acid mimetic that has been shown to have excellenthybridization properties, is referred to as a peptide nucleic acid(PNA). In PNA compounds, the sugar-backbone of an inhibitory nucleicacid is replaced with an amide containing backbone, for example, anaminoethylglycine backbone. The nucleobases are retained and are bounddirectly or indirectly to aza nitrogen atoms of the amide portion of thebackbone. Representative United States patents that teach thepreparation of PNA compounds comprise, but are not limited to, U.S. Pat.Nos. 5,539,082; 5,714,331; and 5,719,262, each of which is hereinincorporated by reference. Further teaching of PNA compounds can befound in Nielsen et al, Science, 1991, 254, 1497-1500.

Inhibitory nucleic acids can also include one or more nucleobase (oftenreferred to in the art simply as “base”) modifications or substitutions.As used herein, “unmodified” or “natural” nucleobases comprise thepurine bases adenine (A) and guanine (G), and the pyrimidine basesthymine (T), cytosine (C) and uracil (U). Modified nucleobases compriseother synthetic and natural nucleobases such as 5-methylcytosine(5-me-C), 5-hydroxymethyl cytosine, xanthine, hypoxanthine,2-aminoadenine, 6-methyl and other alkyl derivatives of adenine andguanine, 2-propyl and other alkyl derivatives of adenine and guanine,2-thiouracil, 2-thiothymine and 2-thiocytosine, 5-halouracil andcytosine, 5-propynyl uracil and cytosine, 6-azo uracil, cytosine andthymine, 5-uracil (pseudo-uracil), 4-thiouracil, 8-halo, 8-amino,8-thiol, 8-thioalkyl, 8-hydroxyl and other 8-substituted adenines andguanines, 5-halo particularly 5-bromo, 5-trifluoromethyl and other5-substituted uracils and cytosines, 7-methylquanine and7-methyladenine, 8-azaguanine and 8-azaadenine, 7-deazaguanine and7-deazaadenine and 3-deazaguanine and 3-deazaadenine.

Further, nucleobases comprise those disclosed in U.S. Pat. No.3,687,808, those disclosed in ‘The Concise Encyclopedia of PolymerScience And Engineering’, pages 858-859, Kroschwitz, J. I., ed. JohnWiley & Sons, 1990, those disclosed by Englisch et al., AngewandleChemie, International Edition’, 1991, 30, page 613, and those disclosedby Sanghvi, Y. S., Chapter 15, Antisense Research and Applications',pages 289-302, Crooke, S. T. and Lebleu, B. ea., CRC Press, 1993.Certain of these nucleobases are particularly useful for increasing thebinding affinity of the oligomeric compounds of the invention. Theseinclude 5-substituted pyrimidines, 6-azapyrimidines and N-2, N-6 and 0-6substituted purines, comprising 2-aminopropyladenine, 5-propynyluraciland 5-propynylcytosine. 5-methylcytosine substitutions have been shownto increase nucleic acid duplex stability by 0.6-1.2<0>C (Sanghvi, Y.S., Crooke, S. T. and Lebleu, B., eds, ‘Antisense Research andApplications’, CRC Press, Boca Raton, 1993, pp. 276-278) and arepresently preferred base substitutions, even more particularly whencombined with 2′-O-methoxyethyl sugar modifications. Modifiednucleobases are described in U.S. Pat. Nos. 3,687,808, as well as4,845,205; 5,130,302; 5,134,066; 5,175,273; 5,367,066; 5,432,272;5,457,187; 5,459,255; 5,484,908; 5,502,177; 5,525,711; 5,552,540;5,587,469; 5,596,091; 5,614,617; 5,750,692, and 5,681,941, each of whichis herein incorporated by reference.

In some embodiments, the inhibitory nucleic acids are chemically linkedto one or more moieties or conjugates that enhance the activity,cellular distribution, or cellular uptake of the inhibitory nucleicacid. Such moieties comprise but are not limited to, lipid moieties suchas a cholesterol moiety (Letsinger et al., Proc. Natl. Acad. Sci. USA,1989, 86, 6553-6556), cholic acid (Manoharan et al., Bioorg. Med. Chem.Let., 1994, 4, 1053-1060), a thioether, e.g., hexyl-S-tritylthiol(Manoharan et al, Ann. N.Y. Acad. Sci., 1992, 660, 306-309; Manoharan etal., Bioorg. Med. Chem. Let., 1993, 3, 2765-2770), a thiocholesterol(Oberhauser et al., Nucl. Acids Res., 1992, 20, 533-538), an aliphaticchain, e.g., dodecandiol or undecyl residues (Kabanov et al., FEBSLett., 1990, 259, 327-330; Svinarchuk et al., Biochimie, 1993, 75,49-54), a phospholipid, e.g., di-hexadecyl-rac-glycerol ortriethylammonium 1,2-di-O-hexadecyl-rac-glycero-3-H-phosphonate(Manoharan et al., Tetrahedron Lett., 1995, 36, 3651-3654; Shea et al.,Nucl. Acids Res., 1990, 18, 3777-3783), a polyamine or a polyethyleneglycol chain (Mancharan et al., Nucleosides & Nucleotides, 1995, 14,969-973), or adamantane acetic acid (Manoharan et al., TetrahedronLett., 1995, 36, 3651-3654), a palmityl moiety (Mishra et al., Biochim.Biophys. Acta, 1995, 1264, 229-237), or an octadecylamine orhexylamino-carbonyl-t oxycholesterol moiety (Crooke et al., J.Pharmacol. Exp. Ther., 1996, 277, 923-937). See also U.S. Pat. Nos.4,828,979; 4,948,882; 5,218,105; 5,525,465; 5,541,313; 5,545,730;5,552,538; 5,578,717, 5,580,731; 5,580,731; 5,591,584; 5,109,124;5,118,802; 5,138,045; 5,414,077; 5,486,603; 5,512,439; 5,578,718;5,608,046; 4,587,044; 4,605,735; 4,667,025; 4,762,779; 4,789,737;4,824,941; 4,835,263; 4,876,335; 4,904,582; 4,958,013; 5,082,830;5,112,963; 5,214,136; 5,082,830; 5,112,963; 5,214,136; 5,245,022;5,254,469; 5,258,506; 5,262,536; 5,272,250; 5,292,873; 5,317,098;5,371,241, 5,391,723; 5,416,203, 5,451,463; 5,510,475; 5,512,667;5,514,785; 5,565,552; 5,567,810; 5,574,142; 5,585,481; 5,587,371;5,595,726; 5,597,696; 5,599,923; 5,599,928 and 5,688,941, each of whichis herein incorporated by reference.

These moieties or conjugates can include conjugate groups covalentlybound to functional groups such as primary or secondary hydroxyl groups.Conjugate groups of the invention include intercalators, reportermolecules, polyamines, polyamides, polyethylene glycols, polyethers,groups that enhance the pharmacodynamic properties of oligomers, andgroups that enhance the pharmacokinetic properties of oligomers. Typicalconjugate groups include cholesterols, lipids, phospholipids, biotin,phenazine, folate, phenanthridine, anthraquinone, acridine,fluoresceins, rhodamines, coumarins, and dyes. Groups that enhance thepharmacodynamic properties, in the context of this invention, includegroups that improve uptake, enhance resistance to degradation, and/orstrengthen sequence-specific hybridization with the target nucleic acid.Groups that enhance the pharmacokinetic properties, in the context ofthis invention, include groups that improve uptake, distribution,metabolism or excretion of the compounds of the present invention.Representative conjugate groups are disclosed in International PatentApplication No. PCT/US92/09196, filed Oct. 23, 1992, and U.S. Pat. No.6,287,860, which are incorporated herein by reference. Conjugatemoieties include, but are not limited to, lipid moieties such as acholesterol moiety, cholic acid, a thioether, e.g., hexyl-5-tritylthiol,a thiocholesterol, an aliphatic chain, e.g., dodecandiol or undecylresidues, a phospholipid, e.g., di-hexadecyl-rac-glycerol ortriethylammonium 1,2-di-O-hexadecyl-rac-glycero-3-H-phosphonate, apolyamine or a polyethylene glycol chain, or adamantane acetic acid, apalmityl moiety, or an octadecylamine or hexylamino-carbonyl-oxycholesterol moiety. See, e.g., U.S. Pat. Nos. 4,828,979; 4,948,882;5,218,105; 5,525,465; 5,541,313; 5,545,730; 5,552,538; 5,578,717,5,580,731; 5,580,731; 5,591,584; 5,109,124; 5,118,802; 5,138,045;5,414,077; 5,486,603; 5,512,439; 5,578,718; 5,608,046; 4,587,044;4,605,735; 4,667,025; 4,762,779; 4,789,737; 4,824,941; 4,835,263;4,876,335; 4,904,582; 4,958,013; 5,082,830; 5,112,963; 5,214,136;5,082,830; 5,112,963; 5,214,136; 5,245,022; 5,254,469; 5,258,506;5,262,536; 5,272,250; 5,292,873; 5,317,098; 5,371,241, 5,391,723;5,416,203, 5,451,463; 5,510,475; 5,512,667; 5,514,785; 5,565,552;5,567,810; 5,574,142; 5,585,481; 5,587,371; 5,595,726; 5,597,696;5,599,923; 5,599,928 and 5,688,941.

Locked Nucleic Acids (LNAs)

In some embodiments, the modified inhibitory nucleic acids (includingASOs) used in the methods described herein comprise locked nucleic acid(LNA) molecules, e.g., including [alpha]-L-LNAs. LNAs compriseribonucleic acid analogues wherein the ribose ring is “locked” by amethylene bridge between the 2′-oxgygen and the 4′-carbon —i.e.,inhibitory nucleic acids containing at least one LNA monomer, that is,one 2′-O,4′-C-methylene-β-D-ribofuranosyl nucleotide. LNA bases formstandard Watson-Crick base pairs but the locked configuration increasesthe rate and stability of the basepairing reaction (Jepsen et al.,Oligonucleotides, 14, 130-146 (2004)). LNAs also have increased affinityto base pair with RNA as compared to DNA. These properties render LNAsespecially useful as probes for fluorescence in situ hybridization(FISH) and comparative genomic hybridization, as knockdown tools formiRNAs, and as antisense oligonucleotides to target mRNAs or other RNAs,e.g., RNAs as described herein.

The LNA molecules can include molecules comprising 10-30, e.g., 12-24,e.g., 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27,28, 29, or 30 nucleotides in each strand, wherein one of the strands issubstantially identical, e.g., at least 80% (or more, e.g., 85%, 90%,95%, or 100%) identical, e.g., having 3, 2, 1, or 0 mismatchednucleotide(s), to a target region in the RNA. The LNA molecules can bechemically synthesized using methods known in the art.

The LNA molecules can be designed using any method known in the art; anumber of algorithms are known, and are commercially available (e.g., onthe internet, for example at exiqon.com). See, e.g., You et al., Nuc.Acids. Res. 34:e60 (2006); McTigue et al., Biochemistry 43:5388-405(2004); and Levin et al., Nuc. Acids. Res. 34:e142 (2006). For example,“gene walk” methods, similar to those used to design antisense oligos,can be used to optimize the inhibitory activity of the LNA; for example,a series of inhibitory nucleic acids of 10-30 nucleotides spanning thelength of a target RNA can be prepared, followed by testing foractivity. Optionally, gaps, e.g., of 5-10 nucleotides or more, can beleft between the LNAs to reduce the number of inhibitory nucleic acidssynthesized and tested. GC content is preferably between about 30-60%.General guidelines for designing LNAs are known in the art; for example,LNA sequences will bind very tightly to other LNA sequences, so it ispreferable to avoid significant complementarity within an LNA.Contiguous runs of more than four LNA residues, should be avoided wherepossible (for example, it may not be possible with very short (e.g.,about 9-10 nt) inhibitory nucleic acids). In some embodiments, the LNAsare xylo-LNAs.

For additional information regarding LNAs see U.S. Pat. Nos. 6,268,490;6,734,291; 6,770,748; 6,794,499; 7,034,133; 7,053,207; 7,060,809;7,084,125; and 7,572,582; and U.S. Pre-Grant Pub. Nos. 20100267018;20100261175; and 20100035968; Koshkin et al. Tetrahedron 54, 3607-3630(1998); Obika et al. Tetrahedron Lett. 39, 5401-5404 (1998); Jepsen etal., Oligonucleotides 14:130-146 (2004); Kauppinen et al., Drug Disc.Today 2(3):287-290 (2005); and Ponting et al., Cell 136(4):629-641(2009), and references cited therein.

Making and Using Inhibitory Nucleic Acids

The nucleic acid sequences used to practice the methods describedherein, whether RNA, cDNA, genomic DNA, vectors, viruses or hybridsthereof, can be isolated from a variety of sources, geneticallyengineered, amplified, and/or expressed/generated recombinantly.Recombinant nucleic acid sequences can be individually isolated orcloned and tested for a desired activity. Any recombinant expressionsystem can be used, including e.g. in vitro, bacterial, fungal,mammalian, yeast, insect or plant cell expression systems.

Nucleic acid sequences of the invention can be inserted into deliveryvectors and expressed from transcription units within the vectors. Therecombinant vectors can be DNA plasmids or viral vectors. Generation ofthe vector construct can be accomplished using any suitable geneticengineering techniques well known in the art, including, withoutlimitation, the standard techniques of PCR, oligonucleotide synthesis,restriction endonuclease digestion, ligation, transformation, plasmidpurification, and DNA sequencing, for example as described in Sambrooket al. Molecular Cloning: A Laboratory Manual. (1989)), Coffin et al.(Retroviruses. (1997)) and “RNA Viruses: A Practical Approach” (Alan J.Cann, Ed., Oxford University Press, (2000)). As will be apparent to oneof ordinary skill in the art, a variety of suitable vectors areavailable for transferring nucleic acids of the invention into cells.The selection of an appropriate vector to deliver nucleic acids andoptimization of the conditions for insertion of the selected expressionvector into the cell, are within the scope of one of ordinary skill inthe art without the need for undue experimentation. Viral vectorscomprise a nucleotide sequence having sequences for the production ofrecombinant virus in a packaging cell. Viral vectors expressing nucleicacids of the invention can be constructed based on viral backbonesincluding, but not limited to, a retrovirus, lentivirus, adenovirus,adeno-associated virus, pox virus or alphavirus. The recombinant vectorscapable of expressing the nucleic acids of the invention can bedelivered as described herein, and persist in target cells (e.g., stabletransformants).

Nucleic acid sequences used to practice this invention can besynthesized in vitro by well-known chemical synthesis techniques, asdescribed in, e.g., Adams (1983) J. Am. Chem. Soc. 105:661; Belousov(1997) Nucleic Acids Res. 25:3440-3444; Frenkel (1995) Free Radic. Biol.Med. 19:373-380; Blommers (1994) Biochemistry 33:7886-7896; Narang(1979) Meth. Enzymol. 68:90; Brown (1979) Meth. Enzymol. 68:109;Beaucage (1981) Tetra. Lett. 22:1859; U.S. Pat. No. 4,458,066.

Nucleic acid sequences of the invention can be stabilized againstnucleolytic degradation such as by the incorporation of a modification,e.g., a nucleotide modification. For example, nucleic acid sequences ofthe invention includes a phosphorothioate at least the first, second, orthird internucleotide linkage at the 5′ or 3′ end of the nucleotidesequence. As another example, the nucleic acid sequence can include a2′-modified nucleotide, e.g., a 2′-deoxy, 2′-deoxy-2′-fluoro,2′-O-methyl, 2′-O-methoxyethyl (2′-O-MOE), 2′-O-aminopropyl (2′-O-AP),2′-O-dimethylaminoethyl (2′-O-DMAOE), 2′-O-dimethylaminopropyl(2′-O-DMAP), 2′-O-dimethylaminoethyloxyethyl (2′-O-DMAEOE), or2′-O-N-methylacetamido (2′-O-NMA). As another example, the nucleic acidsequence can include at least one 2′-O-methyl-modified nucleotide, andin some embodiments, all of the nucleotides include a 2′-O-methylmodification. In some embodiments, the nucleic acids are “locked,” i.e.,comprise nucleic acid analogues in which the ribose ring is “locked” bya methylene bridge connecting the 2′-O atom and the 4′-C atom (see,e.g., Kaupinnen et al., Drug Disc. Today 2(3):287-290 (2005); Koshkin etal., J. Am. Chem. Soc., 120(50):13252-13253 (1998)). For additionalmodifications see US 20100004320, US 20090298916, and US 20090143326.

Techniques for the manipulation of nucleic acids used to practice thisinvention, such as, e.g., subcloning, labeling probes (e.g.,random-primer labeling using Klenow polymerase, nick translation,amplification), sequencing, hybridization and the like are welldescribed in the scientific and patent literature, see, e.g., Sambrooket al., Molecular Cloning; A Laboratory Manual 3d ed. (2001); CurrentProtocols in Molecular Biology, Ausubel et al., eds. (John Wiley & Sons,Inc., New York 2010); Kriegler, Gene Transfer and Expression: ALaboratory Manual (1990); Laboratory Techniques In Biochemistry AndMolecular Biology: Hybridization With Nucleic Acid Probes, Part I.Theory and Nucleic Acid Preparation, Tijssen, ed. Elsevier, N.Y. (1993).

Pharmaceutical Compositions

The methods described herein can include the administration ofpharmaceutical compositions and formulations comprising an inhibitorynucleic acid that targets PAR-TERRA RNA and other chromosome-specificTERRA RNAs.

In some embodiments, the compositions are formulated with apharmaceutically acceptable carrier. The pharmaceutical compositions andformulations can be administered parenterally, topically, orally or bylocal administration, such as by aerosol or transdermally. Thepharmaceutical compositions can be formulated in any way and can beadministered in a variety of unit dosage forms depending upon thecondition or disease and the degree of illness, the general medicalcondition of each patient, the resulting preferred method ofadministration and the like. Details on techniques for formulation andadministration of pharmaceuticals are well described in the scientificand patent literature, see, e.g., Remington: The Science and Practice ofPharmacy, 21st ed., 2005.

The inhibitory nucleic acids can be administered alone or as a componentof a pharmaceutical formulation (composition). The compounds may beformulated for administration, in any convenient way for use in human orveterinary medicine. Wetting agents, emulsifiers and lubricants, such assodium lauryl sulfate and magnesium stearate, as well as coloringagents, release agents, coating agents, sweetening, flavoring andperfuming agents, preservatives and antioxidants can also be present inthe compositions.

Formulations of the compositions of the invention include those suitablefor intradermal, inhalation, oral/nasal, topical, parenteral, rectal,and/or intravaginal administration. The formulations may conveniently bepresented in unit dosage form and may be prepared by any methods wellknown in the art of pharmacy. The amount of active ingredient (e.g.,nucleic acid sequences of this invention) which can be combined with acarrier material to produce a single dosage form will vary dependingupon the host being treated, the particular mode of administration,e.g., intradermal or inhalation. The amount of active ingredient whichcan be combined with a carrier material to produce a single dosage formwill generally be that amount of the compound which produces atherapeutic effect, e.g., an antigen specific T cell or humoralresponse.

Pharmaceutical formulations can be prepared according to any methodknown to the art for the manufacture of pharmaceuticals. Such drugs cancontain sweetening agents, flavoring agents, coloring agents andpreserving agents. A formulation can be admixtured with nontoxicpharmaceutically acceptable excipients which are suitable formanufacture. Formulations may comprise one or more diluents,emulsifiers, preservatives, buffers, excipients, etc. and may beprovided in such forms as liquids, powders, emulsions, lyophilizedpowders, sprays, creams, lotions, controlled release formulations,tablets, pills, gels, on patches, in implants, etc.

Pharmaceutical formulations for oral administration can be formulatedusing pharmaceutically acceptable carriers well known in the art inappropriate and suitable dosages. Such carriers enable thepharmaceuticals to be formulated in unit dosage forms as tablets, pills,powder, dragees, capsules, liquids, lozenges, gels, syrups, slurries,suspensions, etc., suitable for ingestion by the patient. Pharmaceuticalpreparations for oral use can be formulated as a solid excipient,optionally grinding a resulting mixture, and processing the mixture ofgranules, after adding suitable additional compounds, if desired, toobtain tablets or dragee cores. Suitable solid excipients arecarbohydrate or protein fillers include, e.g., sugars, includinglactose, sucrose, mannitol, or sorbitol; starch from corn, wheat, rice,potato, or other plants; cellulose such as methyl cellulose,hydroxypropylmethyl-cellulose, or sodium carboxy-methylcellulose; andgums including arabic and tragacanth; and proteins, e.g., gelatin andcollagen. Disintegrating or solubilizing agents may be added, such asthe cross-linked polyvinyl pyrrolidone, agar, alginic acid, or a saltthereof, such as sodium alginate. Push-fit capsules can contain activeagents mixed with a filler or binders such as lactose or starches,lubricants such as talc or magnesium stearate, and, optionally,stabilizers. In soft capsules, the active agents can be dissolved orsuspended in suitable liquids, such as fatty oils, liquid paraffin, orliquid polyethylene glycol with or without stabilizers.

Aqueous suspensions can contain an active agent (e.g., nucleic acidsequences of the invention) in admixture with excipients suitable forthe manufacture of aqueous suspensions, e.g., for aqueous intradermalinjections. Such excipients include a suspending agent, such as sodiumcarboxymethylcellulose, methylcellulose, hydroxypropylmethylcellulose,sodium alginate, polyvinylpyrrolidone, gum tragacanth and gum acacia,and dispersing or wetting agents such as a naturally occurringphosphatide (e.g., lecithin), a condensation product of an alkyleneoxide with a fatty acid (e.g., polyoxyethylene stearate), a condensationproduct of ethylene oxide with a long chain aliphatic alcohol (e.g.,heptadecaethylene oxycetanol), a condensation product of ethylene oxidewith a partial ester derived from a fatty acid and a hexitol (e.g.,polyoxyethylene sorbitol mono-oleate), or a condensation product ofethylene oxide with a partial ester derived from fatty acid and ahexitol anhydride (e.g., polyoxyethylene sorbitan mono-oleate). Theaqueous suspension can also contain one or more preservatives such asethyl or n-propyl p-hydroxybenzoate, one or more coloring agents, one ormore flavoring agents and one or more sweetening agents, such assucrose, aspartame or saccharin. Formulations can be adjusted forosmolarity.

In some embodiments, oil-based pharmaceuticals are used foradministration of nucleic acid sequences of the invention. Oil-basedsuspensions can be formulated by suspending an active agent in avegetable oil, such as arachis oil, olive oil, sesame oil or coconutoil, or in a mineral oil such as liquid paraffin; or a mixture of these.See e.g., U.S. Pat. No. 5,716,928 describing using essential oils oressential oil components for increasing bioavailability and reducinginter- and intra-individual variability of orally administeredhydrophobic pharmaceutical compounds (see also U.S. Pat. No. 5,858,401).The oil suspensions can contain a thickening agent, such as beeswax,hard paraffin or cetyl alcohol. Sweetening agents can be added toprovide a palatable oral preparation, such as glycerol, sorbitol orsucrose. These formulations can be preserved by the addition of anantioxidant such as ascorbic acid. As an example of an injectable oilvehicle, see Minto (1997) J. Pharmacol. Exp. Ther. 281:93-102.

Pharmaceutical formulations can also be in the form of oil-in-wateremulsions. The oily phase can be a vegetable oil or a mineral oil,described above, or a mixture of these. Suitable emulsifying agentsinclude naturally-occurring gums, such as gum acacia and gum tragacanth,naturally occurring phosphatides, such as soybean lecithin, esters orpartial esters derived from fatty acids and hexitol anhydrides, such assorbitan mono-oleate, and condensation products of these partial esterswith ethylene oxide, such as polyoxyethylene sorbitan mono-oleate. Theemulsion can also contain sweetening agents and flavoring agents, as inthe formulation of syrups and elixirs. Such formulations can alsocontain a demulcent, a preservative, or a coloring agent. In alternativeembodiments, these injectable oil-in-water emulsions of the inventioncomprise a paraffin oil, a sorbitan monooleate, an ethoxylated sorbitanmonooleate and/or an ethoxylated sorbitan trioleate.

The pharmaceutical compounds can also be administered by in intranasal,intraocular and intravaginal routes including suppositories,insufflation, powders and aerosol formulations (for examples of steroidinhalants, see e.g., Rohatagi (1995) J. Clin. Pharmacol. 35:1187-1193;Tjwa (1995) Ann. Allergy Asthma Immunol. 75:107-111). Suppositoriesformulations can be prepared by mixing the drug with a suitablenon-irritating excipient which is solid at ordinary temperatures butliquid at body temperatures and will therefore melt in the body torelease the drug. Such materials are cocoa butter and polyethyleneglycols.

In some embodiments, the pharmaceutical compounds can be deliveredtransdermally, by a topical route, formulated as applicator sticks,solutions, suspensions, emulsions, gels, creams, ointments, pastes,jellies, paints, powders, and aerosols.

In some embodiments, the pharmaceutical compounds can also be deliveredas microspheres for slow release in the body. For example, microspherescan be administered via intradermal injection of drug which slowlyrelease subcutaneously; see Rao (1995) J. Biomater Sci. Polym. Ed.7:623-645; as biodegradable and injectable gel formulations, see, e.g.,Gao (1995) Pharm. Res. 12:857-863 (1995); or, as microspheres for oraladministration, see, e.g., Eyles (1997) J. Pharm. Pharmacol. 49:669-674.

In some embodiments, the pharmaceutical compounds can be parenterallyadministered, such as by intravenous (IV) administration oradministration into a body cavity or lumen of an organ. Theseformulations can comprise a solution of active agent dissolved in apharmaceutically acceptable carrier. Acceptable vehicles and solventsthat can be employed are water and Ringer's solution, an isotonic sodiumchloride. In addition, sterile fixed oils can be employed as a solventor suspending medium. For this purpose any bland fixed oil can beemployed including synthetic mono- or diglycerides. In addition, fattyacids such as oleic acid can likewise be used in the preparation ofinjectables. These solutions are sterile and generally free ofundesirable matter. These formulations may be sterilized byconventional, well known sterilization techniques. The formulations maycontain pharmaceutically acceptable auxiliary substances as required toapproximate physiological conditions such as pH adjusting and bufferingagents, toxicity adjusting agents, e.g., sodium acetate, sodiumchloride, potassium chloride, calcium chloride, sodium lactate and thelike. The concentration of active agent in these formulations can varywidely, and will be selected primarily based on fluid volumes,viscosities, body weight, and the like, in accordance with theparticular mode of administration selected and the patient's needs. ForIV administration, the formulation can be a sterile injectablepreparation, such as a sterile injectable aqueous or oleaginoussuspension. This suspension can be formulated using those suitabledispersing or wetting agents and suspending agents. The sterileinjectable preparation can also be a suspension in a nontoxicparenterally-acceptable diluent or solvent, such as a solution of1,3-butanediol. The administration can be by bolus or continuousinfusion (e.g., substantially uninterrupted introduction into a bloodvessel for a specified period of time).

In some embodiments, the pharmaceutical compounds and formulations canbe lyophilized. Stable lyophilized formulations comprising an inhibitorynucleic acid can be made by lyophilizing a solution comprising apharmaceutical of the invention and a bulking agent, e.g., mannitol,trehalose, raffinose, and sucrose or mixtures thereof. A process forpreparing a stable lyophilized formulation can include lyophilizing asolution about 2.5 mg/mL protein, about 15 mg/mL sucrose, about 19 mg/mLNaCl, and a sodium citrate buffer having a pH greater than 5.5 but lessthan 6.5. See, e.g., U.S. 20040028670.

The compositions and formulations can be delivered by the use ofliposomes. By using liposomes, particularly where the liposome surfacecarries ligands specific for target cells, or are otherwisepreferentially directed to a specific organ, one can focus the deliveryof the active agent into target cells in vivo. See, e.g., U.S. Pat. Nos.6,063,400; 6,007,839; Al-Muhammed (1996) J. Microencapsul. 13:293-306;Chonn (1995) Curr. Opin. Biotechnol. 6:698-708; Ostro (1989) Am. J.Hosp. Pharm. 46:1576-1587. As used in the present invention, the term“liposome” means a vesicle composed of amphiphilic lipids arranged in abilayer or bilayers. Liposomes are unilamellar or multilamellar vesiclesthat have a membrane formed from a lipophilic material and an aqueousinterior that contains the composition to be delivered. Cationicliposomes are positively charged liposomes that are believed to interactwith negatively charged DNA molecules to form a stable complex.Liposomes that are pH-sensitive or negatively-charged are believed toentrap DNA rather than complex with it. Both cationic and noncationicliposomes have been used to deliver DNA to cells.

Liposomes can also include “sterically stabilized” liposomes, i.e.,liposomes comprising one or more specialized lipids. When incorporatedinto liposomes, these specialized lipids result in liposomes withenhanced circulation lifetimes relative to liposomes lacking suchspecialized lipids. Examples of sterically stabilized liposomes arethose in which part of the vesicle-forming lipid portion of the liposomecomprises one or more glycolipids or is derivatized with one or morehydrophilic polymers, such as a polyethylene glycol (PEG) moiety.Liposomes and their uses are further described in U.S. Pat. No.6,287,860.

The formulations of the invention can be administered for prophylacticand/or therapeutic treatments. In some embodiments, for therapeuticapplications, compositions are administered to a subject who is need ofreduced triglyceride levels, or who is at risk of or has a disorderdescribed herein, in an amount sufficient to cure, alleviate orpartially arrest the clinical manifestations of the disorder or itscomplications; this can be called a therapeutically effective amount.For example, in some embodiments, pharmaceutical compositions of theinvention are administered in an amount sufficient to decrease serumlevels of triglycerides in the subject.

The amount of pharmaceutical composition adequate to accomplish this isa therapeutically effective dose. The dosage schedule and amountseffective for this use, i.e., the dosing regimen, will depend upon avariety of factors, including the stage of the disease or condition, theseverity of the disease or condition, the general state of the patient'shealth, the patient's physical status, age and the like. In calculatingthe dosage regimen for a patient, the mode of administration also istaken into consideration.

The dosage regimen also takes into consideration pharmacokineticsparameters well known in the art, i.e., the active agents' rate ofabsorption, bioavailability, metabolism, clearance, and the like (see,e.g., Hidalgo-Aragones (1996) J. Steroid Biochem. Mol. Biol. 58:611-617;Groning (1996) Pharmazie 51:337-341; Fotherby (1996) Contraception54:59-69; Johnson (1995) J. Pharm. Sci. 84:1144-1146; Rohatagi (1995)Pharmazie 50:610-613; Brophy (1983) Eur. J. Clin. Pharmacol. 24:103-108;Remington: The Science and Practice of Pharmacy, 21st ed., 2005). Thestate of the art allows the clinician to determine the dosage regimenfor each individual patient, active agent and disease or conditiontreated. Guidelines provided for similar compositions used aspharmaceuticals can be used as guidance to determine the dosageregiment, i.e., dose schedule and dosage levels, administered practicingthe methods of the invention are correct and appropriate.

Single or multiple administrations of formulations can be givendepending on for example: the dosage and frequency as required andtolerated by the patient, the degree and amount of therapeutic effectgenerated after each administration (e.g., effect on tumor size orgrowth), and the like. The formulations should provide a sufficientquantity of active agent to effectively treat, prevent or ameliorateconditions, diseases or symptoms.

In alternative embodiments, pharmaceutical formulations for oraladministration are in a daily amount of between about 1 to 100 or moremg per kilogram of body weight per day. Lower dosages can be used, incontrast to administration orally, into the blood stream, into a bodycavity or into a lumen of an organ. Substantially higher dosages can beused in topical or oral administration or administering by powders,spray or inhalation. Actual methods for preparing parenterally ornon-parenterally administrable formulations will be known or apparent tothose skilled in the art and are described in more detail in suchpublications as Remington: The Science and Practice of Pharmacy, 21sted., 2005.

Various studies have reported successful mammalian dosing usingcomplementary nucleic acid sequences. For example, Esau C., et al.,(2006) Cell Metabolism, 3(2):87-98 reported dosing of normal mice withintraperitoneal doses of miR-122 antisense oligonucleotide ranging from12.5 to 75 mg/kg twice weekly for 4 weeks. The mice appeared healthy andnormal at the end of treatment, with no loss of body weight or reducedfood intake. Plasma transaminase levels were in the normal range (AST ¾45, ALT ¾ 35) for all doses with the exception of the 75 mg/kg dose ofmiR-122 ASO, which showed a very mild increase in ALT and AST levels.They concluded that 50 mg/kg was an effective, non-toxic dose. Anotherstudy by Krützfeldt J., et al., (2005) Nature 438, 685-689, injectedanatgomirs to silence miR-122 in mice using a total dose of 80, 160 or240 mg per kg body weight. The highest dose resulted in a complete lossof miR-122 signal. In yet another study, locked nucleic acids (“LNAs”)were successfully applied in primates to silence miR-122. Elmen J., etal., (2008) Nature 452, 896-899, report that efficient silencing ofmiR-122 was achieved in primates by three doses of 10 mg kg-1LNA-antimiR, leading to a long-lasting and reversible decrease in totalplasma cholesterol without any evidence for LNA-associated toxicities orhistopathological changes in the study animals.

In some embodiments, the methods described herein can includeco-administration with other drugs or pharmaceuticals, e.g.,compositions for providing cholesterol homeostasis. For example, theinhibitory nucleic acids can be co-administered with drugs for treatingor reducing risk of a disorder described herein.

EXAMPLES

The invention is further described in the following examples, which donot limit the scope of the invention described in the claims.

Experimental Procedures

The following materials and methods were used in the Examples below.

FISH

Cells were cytospun onto glass slides and permeabilized with CSK buffercontaining 0.5% Triton X-100, and fixed in 4% paraformaldehyde. DNAoligos probes for RNA FISH were ordered from Integrated DNATechnologies. For TERRA: (TAACCC)₇-Alexa488-3′ and 5′-Cy5-(TAACCC)₇. ForI4 oligos: I4-47k 5′-Alexa488-TGC ACT GAC GTC CTG TGG CCA CTG GGT GGCGCC AGA GCAT (SEQ ID NO:7); I4-22k: 5′-Cy3-taa tct gaa tat ctg ggc ctccgt gtg cag acc tga ggt t (SEQ ID NO:8); I4 31k: 5′-Cy5-gtc tct gtg tctgtc tct ctg tct ctg tcg cta act cta t (SEQ ID NO:9). DNA oligo probesfor RNA-FISH were mixed at the final concentration 0.5 pmol/μl inhybridization solution (50% formamide, 2×SSC, 2 mg/ml BSA, 10% DextranSulfate-500K). BAC DNA probes and PCR-PAR probes were labeled withfluorophore-dUTP using nick translation, used 1 ng/μl for RNA-FISH and50 ng/μl for DNA FISH at the final concentration in hybridizationbuffer. Hybridization was carried out at 42° C. overnight for RNA FISH.Slides were washed with 2×SSC/50% formamide for 5 min three times at 44°C., and then wash with 2×SSC for 5 min twice at 44° C. For DNA FISH,slides were treated with 0.4 mg/ml RNase A in PBS at 37° C. for 1 hr,washed with PBS, incubated with 0.1 N HCl for 10 min. Slides were washedin PBST (0.2% Tween 20 in 1 × PBS) at RT for 5 mins, and then thedenaturation was carried out in 70% formamide/2×SSC at 80□C for 15 mins.Slides were then washed with PBS, dehydrated with EtOH, and air dried.Hybridization was carried out at 37° C. overnight for DNA FISH, andwashing condition was the same as for RNA-FISH with additional wash in0.1×SSC for 5 min at 44° C. For metaphase spread, cells were incubatedwith 50 ng/ml Colcemid for 2 hr, harvested, washed with PBS, incubatedin cold 0.056M KCl on ice for 30 min and fixed in methanol/acetic acid(3:1). Metaphase spread chromosomes were spread on glass slides, airdried and fixed in 4% formaldehyde.

For pairing assay, digital images were obtained with the Nikon andprocessed using Volocity software (PerkinElmer). In brief, z sectionswere captured at 0.2 μm intervals and 3D images were projected on asingle two-dimensional plane. Distance of Xic-Xic, PAR-PAR (x), and thenuclear areas (A) was analyzed using Volocity software. Only nuclei withtwo resolvable X signals were scored (single dots were excluded).‘Normalized distance’ (ND) is defined as x/d, where d is the nucleardiameter, defined as 2(A/π)^(0.5). PCR-PAR PCR primer pairs were used asfollows:

Primer Sequence SEQ ID NO: P3-F: CTCAGAGCCCAGTGTCAATCAC, 10 P3-R:CACGACCGCTTAGAAGAACCGG 11 P4-F: GAGACGGCCTACCATGTGCTTC, 12 P4-R:GTGAGTGCTGTGAACTCGGCTG 13 P5-F: CAGGGCCTGATTTGGCTTGAAAC 14 P5-R:GAAGAGTAGTCTGACCTCATCTC 15 P6-F: CAGGGCATGATATCCTCTTTGG 16 P6-R:CATTCAATGGTGTTGATGATGGTAC 17 P8-F: GGTTAGAATACAGCGCGGACATTCA 18 P8-R:GTGAATCTCCGAGGCAACTGTC 19

ChIRT-seq Analysis

The PAR-TERRA ChIRT protocol was modified from the original ChIRP andCHART protocols (Chu et al., 2011; Simon et al., 2011) as follows: (i)We used a minimum number of capture probes to reduce off-target effects.(ii) We also increased the shearing size to 0.5-3 kb to preserveintegrity of long noncoding RNAs. (iii) Because we observed that RNaseHis not active in SDS buffer, we used NP40 instead of SDS or N-lauroylsarcosine in the final DNA elution; we used a lower concentration ofNP40 detergent to better preserve RNaseH activity (FIG. 9A).

Specifically, mouse ES cells were grown to 80% confluency and feedercells were removed. 15 millions of cells were spun down and washed withPBS once. Cells were resuspended in 10 ml of PBS and then another 10 mlof 2% of glutaraldehyde were added to fix cells at room temperature for10 min. Crosslinking was then quenched with 0.125 M glycine for 5 min.Cells were than spun down at 2000 g for 5 min at 4° C. Cells were thenwashed with cold PBS and then spun down again. Cell pellets wereimmediately frozen in liquid nitrogen and stored at −80° C. Mouse EScells at embryonic body stages (Day3, Day7) were trypsinized, filteredwith cell strainers (40 μm). The following steps were prepared as thesame as undifferentiated ES cells. Cells were thaw out on ice, and wereresuspened in 1 ml of swelling buffer (0.1 M Tris pH 7.0 10 mM KOAc, 15mM MgOAc, 1% NP40, 1 mM DTT, 1 mM PMSF, 100 U/ml Superase-In[Ambion])for 10 min on ice. Cells were then dounced and pelleted at 2500 g for 5min. Nuclei was further lyzed in nuclear lysis buffer (50 mM Tris pH7.0, 10 mM EDTA, 1% SDS, 1 mM DTT, 1 mM PMSF, protease inhibitor, 100U/ml Superase-In) on ice for 10 min, and sonicated using Bioruptor untilDNA size 0.5-3 kb (it usually takes 1.5 hr and depends on the cellnumbers). Cell lysates were then spun down at 13,000 rpm for 5 min toremove insoluble debris. Cell lysates were then frozen in liquidnitrogen and stored in −80° C. Streptavidin-magnetic Cl (LifeTechnologies) beads were blocked with 500 ng/ul yeast total RNA, and 1mg/ml BSA for 1 hr at 37° C., and respuspended in 1X hybridizationbuffer (1 volume of lysis buffer plus 2 volume of 2X hybridizationbuffer). Cell lysates were diluted in two times volume of 2Xhybridization buffer (750 mM NaCl, 1% SDS, 50 mM Tris pH 7.0, 1 mM EDTA,15% Formamide, 1 mM DTT, PMSF, protease inhibitor, and 100 U/mlSuperase-In), and were preclean with Streptavidin-magnetic Cl beads at37° C. for 1 hr (100 μl of beads for 1 ml lysates). Precleaned lysateswere incubated with pooled probes (100 pmol for 3 ml of diluted celllysates) at 37° C. for 3 hr. Three hundred microliters washed/blocked Clbeads were added per 100 pmol of probes, and the whole reaction wasmixed for another 1 hr at 37° C. DNA probes for ChIRT were ordered fromIntegrated DNA Technologies and labeled with 3′ biotin-TEG. PAR DNAprobe sequences were listed as follows: 36K: gagcgcctcagtgtgcaaatct (SEQID NO:20), 47K: ACTGGGTGGCGCCAGAGCAT(SEQ ID NO:21), 22K:ctccgtgtgcagacctgaggtt (SEQ ID NO:22), 34K: ccctacctaccctccagaga (SEQ IDNO:23), 31K: tctctgtctctgtcgctaac (SEQ ID NO:24). TERRA-AS probesequence: TAACCCTAACCCTAACCCTA (SEQ ID NO:25). TERRA-sense probesequence: TTAGGGTTAGGGTTAGGGTT (SEQ ID NO:26). Beads: biotin-probes:RNA:chromatin adducts were captured by magnets, washed five times at 37°C. for 5 min with wash buffer (2×SSC, 0.5% SDS, 1 mM DTT, 1 mM PMSF),and then washed twice for 5 min at room temperature with 0.1% NP40buffer (150 mM NaCl, 50 mM Tris pH 8.0, 3 mM MgCl₂, 10 mM DTT, 0.1%NP40). DNA was then eluted twice for 20 min in 450 μl of 0.1% NP40buffer with 200 U/ml RNase H (NEB) at room temperature. DNA for no RNaseH controls was eluted in 0.1% NP40 buffer without RNaseH. Eluted DNA wastreated with RNase A (1 mg/ml) at 37° C. for 1 hr, and then was treatedwith proteinase K (1 mg/ml) and supplied addition of SDS to 0.5% atfinal concentration at 55° C. for 16 hr. DNA was extracted withphenol/chloroform using phase lock gel tubes. For pre-RNaseA treatmentcontrol, cell lysates were treated with RNase A at 37° C. overnightbefore hybridization. For RNA elution after hybridization,beads:biotin-probes:RNA:chromatin adducts were washed 5 time in washbuffer, then treated with proteinase K in PK buffer (100 mM NaCl, TrispH 7.0, 1 mM EDTA, 0.5% SDS) at 55° C. for 30 min. Beads suspension wasboiled at 90° C. for 5 min, and then RNA was extracted using TRIzol(Invitrogen). Primer pairs for q-PCR were used as followed: PAR-DNA-F:TGGAGGTTAAACGATTATTTATCTGC (SEQ ID NO:27), PAR-DNA-R:ACGAGTTTCCAAGGTGCTG (SEQ ID NO:28); Hprt-F: CTGCTACTTCAACTCCTGGTGTGC(SEQ ID NO:29), Hprt-R: AGGCGAATTGGGATGTAGCTCAG (SEQ ID NO:30).

PAR-TERRA ChIRT-seq Analysis

PicoGreen (Life Technologies) was used to estimate the concentration ofeluted DNA. Before library construction, equal amount of lambda DNA(0.015 pg of PCR products, ˜250 bp) was added as spike-in control intoeluted DNA samples. The PCR primers sequences for lambda DNA are asfollows: Lambda 5-F, 5′-GCA TAT GTT GTG TTT TAC AG-3′ (SEQ ID NO:31);Lambda 5-R, 5′-GCA ACA AAT TGA TAA GCA-3′ (SEQ ID NO:32). Following theremoval of adaptor sequences and PCR duplicates, paired-end 50 bpsequencing data was aligned to mouse reference genome (GRCm38/mm10 andNCBI37/mm9) using the software Novoalign (v3.00.02) (Li H. (2013)Aligning sequence reads, clone sequences and assembly contigs withBWA-MEM. arXiv:1303.3997v1 [q-bio.GN]). The coverage files weregenerated using R software library SPP software (Kharchenko et al.,2008) with smoothing using 500 bp bins with a 100 bp step size togenerate control-subtracted, normalized read densities. Controls includeinput, sense-ChIRT, and TERRA-ChIRT without RNase H elution (no RNaseH). These data were visualized using IGV software to display all trackswith a mean windowing function and scales indicated in each figure.Other methods to generate normalized coverage files, including thegeneration of conservative enrichment and maximum likelihood estimates,resulted in similar distribution patterns. Scatter plots for correlationanalysis used input—normalized coverage produced by SPP, windowed by 3kb bins and filtered out unenriched bins with an averaged densitysmaller than 4. Peaks were called by MACS (1.4.2)(Zhang et al., 2008)software using normalization to indicated controls (e.g., input, sense,no RNaseH or pre-RNaseA), and filtered by peak length greater than 1 kb.Metagene profiles were produced by software CEAS (0.9.9.7) (Shin et al.,2009) using 2 fold enriched over input wig files and bed files producedby MACS peak calling.

TERRA Knockdown

Mouse ES cells (female, 16.7, cas/mus hybrid) were grown to 70%confluency, and then trypsinized, and feeder cells were removed. A totalof 2×10⁶ mES cells were transfected with LNA gapmer oligos at aconcentration of 2˜8 μM in 100 μl nucleuofector solution using A30program (nucleuofector kits, Lonza). A total of 2 ml offeeders-conditional medium (medium from feeders grown in mES medium for6-18 hr) was added to the cells, and the cells were plated ongelatinized plates. LNA gapmers were designed and synthesized by Exiqonwith modified LNA bases and phosphothiolated backbone modification. TheLNA sequences were as follows: Scr, 5′-CAC GTC TAT ACA CCA C-3′ (SEQ IDNO:4); TERRA, 5′-TAA CCC TAA CCC TAA C-3′ (SEQ ID NO:5); PAR, 5′-TCT CTGTCT CTG TCG C-3′ (SEQ ID NO:6). SV40T transformed MEFs (cas/mus hybrid)were used for TERRA LNA knockdown.

RNA-seq Analysis

Total RNA was isolated using TRIzol (Invitrogen), depleted of DNA byDNase treatment (TURBO DNase, Ambion), depleted of ribosomal RNA(Ribominus Eukaryote Kit v2, Invitrogen), purified greater than 200nucleotides using mirVana RNA extraction kit (Ambion), and fragmented infirst strand synthesis buffer (NEB) containing magenisium at 95° C. for10 min to a median size 150-200 bp. cDNA were reversed transcribed withrandom primers (with Actinomycin D) using Superscript III (Invitrogen)at 50° C. for 30 min. The following steps such as second strandsynthesis, end repaired, dA-tailing, adaptor ligation, USER enzymedigestion, double size selection (0.6x-1.2x AMpure XP beads), andlibrary amplification were performed according to NEBUltra DirectionalRNA library preparation protocol for Illumina (NEB). Sequencing ofpurified libraries was carried out on an Illumina HiSeq instrument forpaired 50 nucleotides reads. After removal of adaptor sequences by TrimGalore, reads were aligned to mouse genomes (GRCm38/mm10 and NCBI37/mm9)using Tophat2. After removal of PCR duplicates, data was analyzed usingeither Cuffdiff 2 (Trapnell et al., 2013). Differential expression wascalled using Cuffdiff 2 with a threshold of q-Value<0.05. Coverage ofRNA-seq was normalized by per million mapped reads as FPM value shown inthe tracks. Allelic RNA-seq analysis was described previously (Simon etal., 2013). Briefly, reads were aligned to allele-specifically to129S1/SvJm (mus) and CAST/EiJ (cas) using Tophat2. All reads mapping togene bodies were summed for cas, mus and comp tracks, and PCR duplicateswere removed. Differential expression between sets of genes in KDsamples was analyzed using R library EdgeR (3.4.2) within by HOMER (4.8)software (Heinz et al., 2010) using function analyseRepeates.pl togenerate count numbers on mus tracks for gene expression on Xi (mus).

Northern Blotting Analysis

DNA sequences for Northern probes were listed as follows:

SEQ ID Probe Sequence NO. TERRA TAACCCTAACCCTAACCCTAACCCTAACCC 33 GAPDHGTAGACCCACGACATACTCAGCACCGGCCTCACCCCATT 34 14-15kaaggccagccgcggttccagacctgcggtgcggccgtgtc 35 14-22ktaatctgaatatctgggcctccgtgtgcagacctgaggtt 36 14-27kttgggggcgtgtctcagagcaggaggggtgtggtctggca 37 14-31kgtctctgtgtctgtctctctgtctctgtcgctaactctat 38 14-34kaaagccaccaggcctctaatccctacctaccctccagaga 39 14-42kcctggagaaatcaagtctgcgaagatccaaaaattaaaat 40 14-47kTGCACTGACGTCCTGTGGCCACTGGGTGGCGCCAGAGCAT 41 14-53kCTGACCACCAGGCTACAGTGTCCTGTAACCGCCAGGCATA 42

All oligo probes were end labeled using T4 polynucleotide kinase. I4-31koligos were used for PAR-TERRA transcripts in FIG. 4B. Total RNA wasextracted using TRIzol followed by acid phenol extraction. Total RNA (5μg) was loaded in each lane. Hybridization was carried out at 42° C.overnight using ULTRAhyb-Oligo hybridization buffer (Ambion).

Quantitative RT-PCR

Total RNA was isolated using TRIzol (Invitrogen), treated with TURBODNase (Ambion), and reverse-transcribed with random primers usingSuperscript III reverse transcriptase (Invitrogen). qRT-PCR was performusing iQ SYBR Green Supermix (Bio-Rad). Expression levels werenormalized to GAPDH levels. Primer pairs were used as follows:

Primer Sequence SEQ ID NO: GAPDH-F CGTCCCGTAGACAAAATGGT 43 GAPDH-RTTGATGGCAACAATCTCCAC 44 Erdr1-F CACAGTGATGTCACCCACGA 45 Erdr1-RGTGAGAATCGCTCCGTCCTG 46 Mid1- GGACGAGAGGGGACAAAGGA 47 intron1-F Mid1-GGTCAAACCTGGACTCTGGCA 48 intron1-R Asmt-F GAAGTGGGACAGGAAGTGAG 49 Asmt-RCGGGAACAGGAAGTGGC 50 Wls-F CCAGTCTAATGGTGACCTGGG 51 Wls-RTGAGAGTCAGCATGCACCAG 52 Tmx3-F TACCGAGGACCACGGACTAA 53 Tmx3-RAATACACGGTGCCTCTTCCG. 54

XIST CHART-seq Analysis

The Xist CHART was modified from the original XIST CHART protocols(Simon et al., 2011). We used 7 oligo probes to target Xist RNA:

Oligo Sequence SEQ ID NO: Xist-503 CAGGTATCCATGGCCCCGATGGGC 55 Xist-1895CTCGGTCTCTCGAATCGGATCCGAC 56 Xist-3322 GAGTTATGGGCACTGCATTTTAGCA 57Xist-5799 TTGTTAAACGCAGGCTAGATCCTGA 58 mXist-1240CGCCATTTTATAGACTTCTGAGCAG 59 mXist-935 CCtaattcttggcgtaactggctcg 60mXist-5651 ATGCTTAGGAAGAGGGACAAATGCA 61

In detail, 20 million cells were crosslinked by with 1% formaldehyde for10 min at room temperature. Crosslinking was then quenched with 0.125 Mglycine for 5 min. After washing 3 times with PBS, crosslinked cellswere re-suspended in 2 ml of sucrose buffer (0.3 M sucrose, 1%Triton-X-100, 10 mM HEPES pH 7.5, 100 mM KOAc, 0.1 mM EGTA), dounced 20times with a tight pestle, and kept on ice for 10 min. The followingsteps were using polystyrene tubes,glass pipettes, and DNA LoBindmicrotubes (Eppendorf) to avoid cell clumps sticking onto the walls oftubes or pipettes. Nuclei were collected by centrifugation at 1,500 gfor 10 min on top of a cushion of 5 ml glycerol buffer (25% glycerol, 10mM HEPES pH7.5, 1 mM EDTA, 0.1 mM EGTA, 100 mM KOAc). Nuclei werefurther crosslinked with 3% formaldehyde for 30 min at room temperature.After washing three times with PBS, nuclei were extracted once with 50mM HEPES pH7.5, 250 mM NaCl, 0.1mM EGTA, 0.5% N-lauroylsarcosine, 0.1%sodium deoxycholate, 5 mM DTT, 100 U m121 SUPERasIN (Invitrogen) for 10min on ice, and centrifuged at 400 g for 5 min at 4 uC. Nuclei wereresuspended in 270 μl of sonication buffer (50 mM HEPES pH 7.5, 75 mMNaCl, 0.1 mM EGTA, 0.5% N-lauroylsarcosine, 0.1% sodium deoxycholate, 5mM DTT, 10 U/ml SUPERasIN, and sonicated in microtubes using Covaris S2sonicator at 10% duty cycle, 200 bursts per cycle, intensity 3 for 5min. The size of chromatin fragments was 0.2˜3 kb. Fragmented chromatinwas subjected to hybridization immediately. Hybridization, washing andelution were performed similarly to TERRA-ChIRP protocol. In brief,beads were blocked by yeast tRNA and BSA. 320 μl of 2X hybridizationbuffer (750 mM NaCl, 1% SDS, 50 mM Tris pH 7.0, 1 mM EDTA, 15%Formamide, 1 mM DTT, PMSF, protease inhibitor, and 100 U/ml Superase-in)was added into 160 μl lysates, and then this 1X hybridization lysate wasprecleaned by 60 μl of blocked beads at room temperature for 1 hr. Afterremoval of the beads, 7 probes (labeled with 3′ biotin-TEG) for Xist RNA(3.66 pmol/per probe) were added into the 1X hybridization lysate andincubate at room temperature for overnight.Beads:biotin-probes:RNA:chromatin adducts were captured by magnets,washed once with 1X hybridization buffer at 37° C. for 10 min, washedfour times at 37° C. for 5 min with wash buffer (2×SSC, 0.5% SDS, 1 mMDTT, 1 mM PMSF), and then washed twice for 5 min at room temperaturewith 0.1% NP40 buffer (150 mM NaCl, 50 mM Tris pH 8.0, 3 mM MgCl₂, 10 mMDTT, 0.1% NP40). DNA was then eluted twice for 20 min in 450 μl of 0.1%NP40 buffer with 200 U/ml RNase H (NEB) at room temperature.

Metagene Analysis

Escapee genes are as previously described (Carrel and Willard, 2005;Yang et al., 2010; Pinter et al., 2012). Xist itself is excluded as anescapee in the metagene analysis. “Repressed” genes are all other geneson the Xi which have an FPKM>1.0 on the Xa. The normalized coveragefiles produced from SPP were used for metagene analysis with CEASsoftware.

Example 1 Identification of Sex-Linked PAR-TERRA Transcripts

RNA fluorescence in situ hybridization (FISH) using TERRA oligo probesshowed that TERRA can be seen, in high-exposure and higher contrastimages, as multiple foci in the nuclei of ES cells (FIG. 1A). Consistentwith our previous report (Zhang et al., 2009), two of the speckles wereespecially prominent. To confirm their colocalization next to the sexchromosomes, we performed serial RNA-DNA FISH using probes from thepseudoautosomal region (PAR) of the sex chromosomes. The PAR representsthe only homologous region between chromosomes (Chr) X and Y. BecausePAR genes are shared between the sex chromosomes, these genes are notsubject to XCI. At the commencement of this project, the most distallymapped X- and Y-linked gene was the PAR gene, Mid1 (Erdr1 and Asmt werepartially assembled in recent months). We obtained two BAC clonesmapping to Mid1—the 15 kb BAC RP24-143B12 and the ˜146 kb RP24-500I4.RP24-500I4 contains several internal telomeric repeats, two of 40 bp andthe third of 314 bp (FIG. 1B). To isolate PAR-specific probes, wesubcloned the BACs, generated unique PCR fragments, and identified a setof unique probes consistenting of P3, P4, P5, P6, and P8 (P34568; FIG.8A,B). Serial RNA-DNA FISH showed that the large TERRA foci indeedmapped to the PAR in ES cells. Quantitation of RNA FISH intensitiesindicated that sex chromosome-associated TERRA RNA accounted for ˜80-90%of total TERRA transcripts and the finer speckles ˜10-20% of detectedsignals (FIG. 1A,C).

Although cytological analysis shows that TERRA RNA localizes to the endsof most, if not all chromosomes (Azzalin et al., 2007; Schoeftner andBlasco, 2008), the origin of TERRA transcription is not fully known.TERRA may be transcribed by all telomeres and retained in cis, or it maybe transcribed by only a few loci but localized in trans to multipledistant sites. A murine transcriptomic study indicates that TERRA issynthesized predominantly from the end of Chr18 (de Silanes et al.,2014). Because sub-telomeric sequences of some chromosomes, includingChr X and Y, have not been fully sequenced or assembled, determiningadditional transcriptional origins for TERRA is possible.

To determine whether the sub-telomeric region of ChrX and ChrY couldcontribute to sex-linked TERRA transcription, we carried out RNA FISH tocompare signals arising from PAR versus TERRA probes. The PAR and TERRARNA clusters looked nearly identical in male and female ES cells (FIG.1D,E), raising the possibility that PAR and TERRA may extend—at least ina fraction of total transcription—as a single long noncoding RNA. OnNorthern blot analyses, an antisense TERRA oligo probe detected a smearof signals from 100 bp to>9kb (FIG. 1F, left panel), consistent withTERRA being of heterogeneous size (Azzalin et al., 2007; de Silanes etal., 2014). However, we observed a dominant species in ES cells of>>9kb, indicating that some TERRA transcripts may originate much furtherupstream in relation to the telomeric repeats. Primer extension using anantisense TERRA oligo probe gave positive amplification by RT-PCR usingPAR-specific primer pairs located at 33, 36, and 39 kb from the end ofBAC RP24-500I4 (FIG. 1F, right panel), demonstrating that PAR-initiatedRNAs is physically contiguous with at least a fraction ofTERRA-containing RNA. We then repeated Northern analysis using sub-BACwalking probes to verify the physical contiguity and observed smears ofhigh molecular weight transcripts similar to those observed with TERRAprobes (FIG. 1F,G). The pattern was especially similar with probes 22k,27k, 34k, 36k, and 47k. Two-color RNA FISH using BAC sub-probes showedthat >90% of large RNA clusters were coincident with TERRA foci in EScells (FIG. 1H) as well as in MEF (FIG. 8C). In MEFs, ˜93% of PAR andTERRA clusters (n=285) localized next to, but generally did not overlapwith, the Xist cloud (FIG. 8D). Henceforth, we refer to the TERRAtranscripts of PAR origin as “PAR-TERRA”, to distinguish them from TERRAtranscripts intrinsic to the TTAGGG repeats and to those originatingwithin sub-telomeric regions of autosomes.

TERRA foci were similarly observed in various human cells (FIG. 8C). Inmice, PAR-TERRA expression showed some strain-specific variation. Forexample, PAR-TERRA signals were detectable with the 22k probe in hybridES and MEF cells of mixed Mus musculus (mus) and Mus castaneus (cas)origin. Using the 22k probe, however, PAR-TERRA could not be detected incells of pure musculus origin (FIG. 8E). This is consistent with the PARbeing variable in sequence between mouse strains (Soriano et al., 1987).As a consequence, the two large TERRA foci were often asymmetric in sizein hybrid 16.7 female ES cells, with Mus musculus cluster being smallerthan the Mus castaneus cluster. In J1 male ES cells, TERRA RNAs on bothChr X and Y were usually smaller, consistent with their being of Musmusculus origin. We conclude that a substantial fraction of sex-linkedTERRA transcripts originates in the pseudoautosomal region (PAR-TERRA).The X-linked origin could have been missed previously (de Silanes etal., 2014) because pseudoautosomal sequences and assembly were notavailable until recently.

Example 2 Mapping Genome-Wide Targets of TERRA and PAR RNA by ChIRT-seq

Although RNA FISH showed that >90% localized in cis to Chr X and Y,additional foci throughout the nucleus were clearly evident (FIG. 1H).Because the probes were PAR-specific, this finding indicated thatPAR-TERRA could diffuse away from the sex chromosomes and possiblylocalize elsewhere in the nucleus. To identify genome-wide targets ofTERRA and PAR RNA, we captured RNA-bound genomic sites by mergingelements of ChIRP (Chu et al., 2011) and CHART (Simon et al., 2011) toachieve high specificity of chromatin pulldown (henceforth “ChIRT”).Because capture probes could potentially interact with DNA rather thanRNA, we included an RNaseH elution step (FIG. 9A). Several DNA-basedcapture probes were designed: (i) TERRA antisense (TERRA-AS), to capturetranscripts containing UUAGGG, (ii) PAR, to capture PAR-TERRAtranscripts, and (iii) TERRA sense (TERRA-S, the reverse complement), tocontrol for background. Slot blot analysis showed that both TERRA-AS and31k-PAR-AS probes captured TERRA RNA after ChIRT, whereas thecorresponding sense probes did not (FIG. 2B). Quantitative PCR indicatedthat PAR sequences were enriched relative to Hprt sequences after ChIRTusing TERRA-AS and PAR-AS probes, but not when the TERRA-S probe wasused (FIG. 2C). The enrichment was dependent on RNaseH treatment and wasabolished by RNase A treatment (FIG. 2D), indicating that the pulldownwas mediated by interaction between the DNA capture probes and RNAtargets.

We then performed deep sequencing of ChIRT-seq pulldowns to identifygenome-wide binding sites. To rule out artifacts due to direct probehybridization to genomic DNA rather than the intended RNA target, wesequenced two critical controls: (i) an RNaseH-control in which RNaseHwas omitted in the elution step, which would in principle precludeelution of RNA-dependent interactions; and (ii) a TERRA-S control, whichwould not hybridize to TERRA RNA but could potentially pull downcontaminating DNA (in addition to any potential antisense-TERRAtranscripts). We collected ES cells on differentiation days 0, 3, 7 (d0,d3, d7) and MEFs for ChIRT-seq. Approximately 30 million 50-bppaired-end reads were obtained for each library. After removing PCRduplicates, >70% of reads uniquely mapped to the mouse genome.Biological replicates showed a high degree of correlation (FIG. 9B,C).

We used MACS software to call statistically significant enrichment peakssignifying genomic binding sites of TERRA and PAR transcripts (FIG. 2E).To call enrichment peaks, we normalized ChIRT reads to (i) inputlibrary, (ii) TERRA-S library, or (iii) no-RNaseH library. The resultswere highly similar with each method of normalization (FIG. 2F), andMACS called a similar number of peaks (FIG. 2E). Major enrichment attelomeric repeat DNA in the TERRA-AS pulldown relative to TERRA-S andno-RNaseH controls provided a validation of our ChIRT method (FIG. 2G).Across the genome, we observed >2,000 peaks of TERRA binding in day 0and day 3 ES cells, 1,800 peaks in d7 differentiating ES cells, and ˜500peaks in MEFs (FIG. 2E). Peaks called in the sense ChIRT samples did notoverlap those called for TERRA or PAR (FIG. 2F), and could representeither background or binding of a putative antisense TERRA transcript.Similarly for PAR RNA, we observed thousands of binding sites in EScells and hundreds in MEFs. Overall, PAR-TERRA binding sites wereenriched in noncoding space, including upstream regulatory regions,introns, and intergenic space, whereas binding in exons was depleted(FIG. 2H,I).

There was considerable overlap between TERRA and PAR ChIRT profiles,with high Pearson's r values in correlation plots for d0 and d3 ES cellsand for MEFs, but not for comparisons to sense-ChIRT controls (FIG. 2F).There was also a high degree of similarity between biological replicates(FIG. 9B,C). In ES cells and MEFs, whole-genome views demonstratedstrongest overall enrichment of both TERRA and PAR RNAs at telomericends, inclusive of sub-telomeric regions—regions with unique sequencethat enabled unambiguous alignment of paired-ends TERRA reads to thespecific chromosome ends (FIG. 3A). Intriguingly, the X-linked RNAscould diffuse to autosomes and bind their telomeric ends (FIG. 3A, PARtrack). In MEFs, the number of TERRA and PAR targets decreased overall,but telomeric ends remained enriched. Control TERRA-S pulldowns did notresemble TERRA-AS pulldowns and showed no significant enrichment attelomeres. These results demonstrated the specificity of the TERRA andPAR pulldowns and argued against artifacts of genomic DNA hybridization.Taken together, our findings demonstrate that telomeric RNAs (i) areindeed produced from the sex chromosomes and (ii) bind both in cis andin trans to their site of synthesis.

Table 1 provides a list of X-linked genes with the highest PAR-TERRAbinding. Top binders are defined as the top quartile in terms ofPAR-TERRA density over the gene. There are 452 active genes in the MEFcell line. Mm10 coordinates are used. Start and stop positions of thegene target, along with the gene name and transcribed strand are shown.RNA-seq FPKM refers to gene expression in frequency per kilobase permillion reads. Mean TERRA coverage over the gene is also shown.

TABLE 1 PAR-TERRA binding sites on the mouse X-chromosome in MEFs Meanof mm10 TERRA assembly Start End gene name FPKM strand coverage chrX169685246 169990797 Mid1 1.39743 + 203.232 chrX 169311530 169320343 Hccs27.2857 − 64.2334 chrX 167207093 167209218 Tmsb4x 454.052 − 24.1368 chrX152336851 152342484 Tspyl2 28.3268 − 13.6914 chrX 166499814 166510478Tceanc 2.77384 − 13.1733 chrX 74270815 74273135 Rpl10 82.0002 + 12.988chrX 74270815 74273135 Snora70 201.378 + 12.988 chrX 74273216 74282333Dnase1l1 1.20938 − 12.9053 chrX 134601285 134607054 Hnrnph2 44.022 +11.0383 chrX 74369218 74373349 Slc10a3 10.7721 − 10.2314 chrX 153498231153501558 Ubqln2 81.5338 + 10.2078 chrX 73673132 73682500 Slc6a857.6063 + 10.028 chrX 168795098 169304435 Arhgap6 1.7761 + 9.93581 chrX134588168 134601005 Gla 10.9353 − 9.78217 chrX 168654117 168673902 Msl37.40496 − 9.71429 chrX 134686518 134697772 Armcx4 9.21521 + 9.7044 chrX152233229 152274354 Kdm5c 101.365 + 9.38192 chrX 101449108 101453541Itgb1bp2 1.18288 + 8.99717 chrX 101429650 101448593 Nono 123.217 +8.59172 chrX 74313032 74320149 Fam50a 52.844 + 8.43926 chrX 9463606894638561 Gspt2 18.0414 + 8.38342 chrX 166457251 166479867 Rab9 44.5493 −8.34863 chrX 74282696 74290151 Taz 20.6918 + 8.29363 chrX 9913612999148991 Efnb1 8.80092 + 8.27655 chrX 152004583 152016295 Ribc1 8.85901− 8.25976 chrX 101254527 101260873 Foxo4 35.4845 + 7.89614 chrX152144267 152225236 Iqsec2 3.55372 + 7.89114 chrX 13281021 13293983Ddx3x 202.014 + 7.79031 chrX 74365717 74368548 Ubl4 27.821 − 7.72789chrX 152001895 152004442 Hsd17b10 58.1856 + 7.67299 chrX 101532734101601789 Taf1 47.7176 + 7.64038 chrX 152016427 152061973 Smc1a57.1067 + 7.62341 chrX 152294827 152327493 2900056M20Rik 1.73042 −7.3519 chrX 150571506 150588149 Apex2 30.0146 − 7.27157 chrX 151803281151935417 Huwe1 161.224 + 7.14147 chrX 20688492 20699877 Cdk16 89.5491 +6.96555 chrX 101404383 101420685 Zmym3 53.5806 − 6.93208 chrX 103560909103623754 Ftx 6.13111 − 6.83641 chrX 134748454 134751419 Armcx6 1.47279− 6.69088 chrX 8061170 8074760 Suv39h1 10.5102 − 6.59123 chrX 109095406109162467 Sh3bgrl 11.0008 + 6.35196 chrX 94535473 94542074 Maged1150.771 − 6.24733 chrX 96096044 96168553 Msn 125.765 + 6.23976 chrX73483634 73495936 Bgn 131.985 + 6.19272 chrX 163909159 163929546 Ap1s222.2628 + 6.12689 chrX 106015699 106022450 Cox7b 81.8186 + 6.02631 chrX74013913 74023936 Irak1 54.9015 − 5.97292 chrX 74013913 74023936 Mir513228310 − 5.97292 chrX 166523006 166585716 Egfl6 2.75673 − 5.89232 chrX101274090 101298934 Med12 16.9166 + 5.74426 chrX 74223460 74246534 Flna119.321 − 5.66957 chrX 139779680 139782353 Ripply1 9.2593 − 5.66923 chrX94188708 94212651 Eif2s3x 13.3916 − 5.62242 chrX 140539528 140600522Tsc22d3 4.01031 − 5.60397 chrX 74254838 74257747 Emd 131.791 + 5.52669chrX 94074630 94123407 Zfx 14.4577 − 5.457 chrX 8138974 81479632900002K06Rik 2.48279 + 5.45401 chrX 8138974 8147963 Rbm3 21.0491 −5.45401 chrX 151047232 151096543 Fgd1 81.8043 + 5.44937 chrX 151047232151096543 Tsr2 23.1259 − 5.44937 chrX 57383347 57393036 Rbmx 15.7016 −5.4457 chrX 73916869 73921944 Naa10 94.1038 − 5.41574 chrX 7429709674304721 Atp6ap1 57.5415 + 5.33398 chrX 100622905 100625907 Pdzd1167.6827 − 5.24963 chrX 166440824 166452543 Trappc2 5.95827 + 5.23482chrX 101640063 101684351 Ogt 82.85 + 5.19793 chrX 74329065 74344689Plxna3 6.81077 + 5.15815 chrX 73716596 73738287 Abcd1 9.8525 + 5.05484chrX 7884243 7894492 Slc35a2 8.80274 + 5.05244 chrX 7919821 7928607 Eras53.4302 − 5.03256 chrX 7919821 7928607 Pcsk1n 14.3249 + 5.03256 chrX73437314 73459029 Haus7 37.766 − 5.03236 chrX 7894518 7899269 Pqbp1101.762 − 4.95918 chrX 140948424 140956711 Psmd10 16.0633 − 4.86065 chrX160502165 160598878 Phka2 10.5119 + 4.82787 chrX 159627407 159975917Sh3kbp1 13.5148 + 4.81242 chrX 9654269 9662983 Dynlt3 8.16616 − 4.77036chrX 60891365 60893430 Sox3 5.66273 − 4.76974 chrX 73778962 73786897Idh3g 186.084 − 4.72145 chrX 142317992 142390535 Acsl4 25.1009 − 4.71533chrX 164373547 164402647 Figf 2.67737 + 4.70077 chrX 7823842 7836503Kcnd1 5.67212 + 4.6921 chrX 163935442 163958666 Zrsr2 32.212 − 4.68589chrX 159372194 159385699 Eif1ax 160.42 + 4.667 chrX 150983132 151017322Gnl3l 79.5544 − 4.63998 chrX 7722248 7728201 Wdr45 57.6147 + 4.57451chrX 7762660 7775202 Tfe3 88.3365 + 4.55791 chrX 101377336 101385624Gjb1 7.30102 + 4.55117 chrX 155213138 155216409 Sat1 31.7602 − 4.49062chrX 164419786 164433915 Piga 14.5205 + 4.46376 chrX 73686182 73716175Bcap31 136.655 − 4.34133 chrX 7728570 7731063 Praf2 45.3074 + 4.32855chrX 136139044 136140437 Bex4 54.8305 + 4.31599 chrX 134585653 134588062Rpl36a 1.66059 + 4.30142 chrX 134804141 134809221 Armcx2 20.2464 −4.29013 chrX 73853779 73880834 L1cam 1.33532 − 4.26027 chrX 5803062758036630 Zic3 68.0886 + 4.24685 chrX 159532667 159593081 A830080D01Rik43.9242 + 4.19267 chrX 7899397 7907652 Timm17b 31.5895 + 4.06939 chrX142853473 142966728 Ammecr1 13.3866 − 4.01434 chrX 164070702 164076049Siah1b 49.6673 − 3.99607 chrX 150806420 150814339 Maged2 15.8384 −3.94816 chrX 100626064 100727271 Kif4 34.3476 + 3.93177 chrX 7509585375130949 Dkc1 136.814 + 3.90988 chrX 75095853 75130949 Mpp1 38.769 −3.90988 chrX 37091833 37110322 Upf3b 39.8344 − 3.88822 chrX 1293687212938541 AA414768 7.27643 + 3.8628 chrX 48411048 48463132 Elf4 16.0656 −3.8481 chrX 7959259 7967910 Gata1 1.29963 − 3.77677 chrX 134308162134362639 Cenpi 16.8886 + 3.77197 chrX 134059348 134086821 Cstf245.5011 + 3.75403 chrX 51003913 51018018 Rap2c 14.0536 − 3.69198 chrX8238667 8252406 Ftsj1 29.4 − 3.68661 # Top 25% of TERRA mean coverageover gene # Total active gene 452

Example 3 PAR-TERRA Localizes in cis and in trans

Although TERRA and PAR ChIRT profiles were very similar, ChIRT analysisrevealed heterogeneity in the telomeric RNAs. Some binding sites weredominated by TERRA RNA (e.g., telomeric ends of Chr 3,4,5, etc), whileothers showed prominent peaks of both TERRA and PAR RNA (FIG. 3). Adistinct PAR-TERRA species was further supported by the nearly identicalChIRT profiles at these sites of overlap (examples shown in FIG. 3B-D),as well as by the above molecular analyses indicating a physicalcontiguity (FIG. 1F,G). PAR-TERRA binding was especially notable at thesubtelomeric end (pseudoautosomal region) of the sex chromosomes (FIG.3B). Mid1, Erdr1, and Asmt are tens of kilobases away from the TERRA DNAsequence [(TTAGGG)n telomeric repeat], but nevertheless demonstratedprominent TERRA peaks. The intronic regions and 3′ end of Mid1 containedsome of the strongest PAR-TERRA peaks in the genome. The gene body ofAsmt was also a strong binding site. By far the strongest peaks werefound in Erdr1, which itself contains two short stretches of (TTAGGG)nrepeats. [Note: It should be emphasized that these TERRA reads could beunambiguously assigned to the Erdr1 repeats—and not telomericrepeats—because pair-end sequencing enabled utilizing the uniquesequence at one end to align the other repetitive end.] These resultsdemonstrated that PAR-TERRA not only localizes in cis to the telomeresof sex chromosomes, but also spreads locally in cis to emcompass genesof the pseudoautosomal region.

Intriguingly, PAR-TERRA also targeted sites in trans. Magnified viewsshowed strong PAR-TERRA binding peaks in the sub-telomeric regions ofChr 2, 9, 13, and 18 (FIG. 3C), and more moderate binding peaks in thesub-telomeric regions of Chr 8 and 19 (FIG. 10). Additionally, PAR-TERRAtargeted internal regions of autosomes. Many binding peaks occurredwithin genes, especially within introns, as exemplified byAbcb10,Uchl1os, and Hes3 (FIG. 3D). PAR-TERRA also targeted internal(TTAGGG)n telomeric repeats which occur at a number of locationsthroughout the genome (one example shown in FIG. 3C; NOTE: The readscould be assigned unambiguously to the internal repeats because ofpair-end sequencing, in which the unique end is used to align the repeatend). We conclude that TERRA and PAR-TERRA transcripts are not confinedto telomeric ends of mouse chromosomes.

Example 4 Epigenomic Regulation by PAR-TERRA

Because PAR-TERRA accounts for the vast majority of TERRA transcripts inthe nucleus and has both X-linked and autosomal targets, we examined itseffect on gene expression on a genome-wide basis. We perturbed PAR-TERRAexpression using knockdown (KD) approaches to avoid undesirableconsequences of genetically deleting telomeres. Neither siRNA nor shRNAresulted in knockdown. On the other hand, single-stranded antisenseoligonucleotide (ASO) locked nucleic acids (LNA) gapmers led tosubstantial depletion of TERRA after 12 hours in ES cells, as shown byNorthern blot analysis (FIG. 4A, 11A) and RNA FISH (FIG. 4B, 11B). ASO'sto TERRA sequences and PAR-specific sequences (31k) achieved similarresults. At optimal LNA concentrations, at least 75-90% of PAR-TERRAwere degraded as quantitated by Northern analysis (FIG. 4A, 11A). RNAFISH showed a substantial depletion of both TERRA and PAR signalsin >86% of nuclei (n=217) after 6 hours (4B, 11B). Notably, treatingwith 31k-PAR gapmers dramatically reduced TERRA RNA FISH signals (FIG.4B). Conversely, administering TERRA gapmers also reduced PAR FISHsignals. These data provide strong support for the idea of a continuousPAR-TERRA transcript and the conclusion that PAR-TERRA accounts for themajority (˜90%) of telomeric transcripts in ES cells.

We then carried out transcriptomic analysis on two biological replicatesof ES cells after 12 hours of PAR-TERRA depletion (FIG. 4, 11C).Analysis of the biological replicates of TERRA-specific and PAR-specificKD's revealed overlapping transcriptomic changes (FIG. 4C). Analysisusing Cuffdiff2 uncovered 126 differentially expressed genes after TERRAKD and 324 after PAR KD in ES cells, among which 56 genes were sharedbetween the TERRA- and PAR-specific KD cells. In MEFs, TERRA KD led to137 significant changes and PAR KD led to 309, among which 36 genes wereshared. Among the shared genes for each cell type, the changes in geneexpression after PAR and TERRA KD were very similar (FIG. 4D). Among 8genes that were shared between female ES cells and MEFs, the expressionchanges were also highly similar (FIG. 4E).

On the other hand, there was a significantly increased probability thatgenes with PAR-TERRA binding sites would be downregulated byPAR-specific KD (FIG. 4F). The probability density function for 565genes with PAR-TERRA binding sites was significantly different from thatfor the 14,724 genes without a PAR-TERRA site (FIG. 4F; KS testP<0.0001). PAR KD resulted in net downregulation of genes with PAR-TERRAbinding sites (left-shift of red distribution). Among all downregulatedgenes after PAR KD, the degree of downregulation was significantly morepronounced for those with PAR-TERRA sites (FIG. 4G, right panels; X²test, P<0.001), possibly indicative of a direct effect on PAR-TERRAtarget genes. In contrast, among the upregulated genes, there was nosignifcant difference between genes with and without a PAR-TERRA site(□² test, P=0.26), suggesting that upregulation may be an indirectconsequence. These data argue that PAR-TERRA RNA exerts a directpositive effect on target genes.

The non-overlapping transcriptomic changes for TERRA-versus PAR-specificknockdowns may suggest the presence of a gene class targeted byTERRA-intrinsic transcripts (containing only UUAGGG repeats), ratherthan by PAR-TERRA. Consistent with this, we observed a number ofautosomal genes—particularly those within sub-telomeric regions—whereTERRA RNA was the predominant bound form (FIG. 5A). At such genes, TERRAKD consistently resulted in gene downregulation, as shown by RNA-seq andconfirmed by RT-qPCR (FIG. 5B). Such autosomal sub-telomeric genes mighttherefore be controlled by TERRA transcripts produced in cis, perhaps inaddition to some contribution from X-linked PAR-TERRA in trans. Weproposed that genes responsible for facioscapulohumeral musculardystrophy (FSHD) can be controlled in this way. The FSHD locus islocated in the subtelomeric region of human Chr4 and contains codinggenes FRG1, FRG2, DUX4, and the macrosatellite repeat, D4Z4. FSHD iscaused by ectopic expression of these genes when the D4Z4 repeatcontracts and becomes “activated”. Thus, PAR-TERRA or Chr4-specificTERRA could be targeted to downregulate the associated subtelomericgenes.

Example 5 PAR-TERRA Protects Pseudoautosomal Genes and Escapees from XCI

ChrX has a large number of PAR-TERRA sites (FIG. 3A). In d0 ES cells,ChrX harbors 84-86 PAR-TERRA sites; in MEFs, ChrX harbors 30-94 sites,with one broad domain at the distal end of ChrX, another around theX-inactivation center, and several additional hotspots in more proximalregions of ChrX (FIG. 5C,D). Intriguingly, PAR-TERRA densities weregreater at escapees than at genes subject to XCI (FIG. 5E; P<0.05).Escapees play important roles in human disease (e.g., XO TurnerSyndrome) and generally have ChrY homologues that render XCI unnecessaryfor these genes (Berletch et al., 2010; Berletch et al., 2011; Deng etal., 2014). Escapee genes are located throughout ChrX, and may occursingly or in clusters (Berletch et al., 2011; Lopes et al., 2011). Inhumans, 15% of X-linked genes escape silencing, whereas approximately adozen or so genes escape XCI in mice (Carrel and Willard, 2005; Yang etal., 2010). Their association with PAR-TERRA raised the possibility thatPAR-TERRA could regulate escape from XCI.

To test this idea, we performed quantitative RT-PCR. Significantly,PAR-TERRA KD resulted in downregulation of all pseudoautosomal genes,including Mid1, Erdr1, and Asmt (FIG. 5F). This downregulation wasconfirmed by RNA-seq analysis and was similar to the downregulation atsub-telomeric regions of autosomes after TERRA KD (FIG. 5A,B). Thus, oneof TERRA's cis functions may be to protect sub-telomeric genes fromposition effects of telomeric heterochromatin. To examine Xi-specificchanges, we performed allele-specific RNA-seq analysis using a hybridMEF clonal cell line in which the Xi is always of Mus musculus originand the Xa is of Mus castaneus origin (Pinter et al., 2012). RNA-seqanalysis confirmed downregulation of PAR genes and additional knownescapees outside of the PAR, such as Shroom4, Kdm6a, 1810030O07Rik, aswell as escapees (Fgf13, Mbtps2, Huwe1, Sept6, Aifm1, and Kif4) observedin our cell line. We concluded that PAR-TERRA promotes expression ofescapee genes on the Xi.

We next investigated the mechanistic relationship between PAR-TERRA andescapees. While escapee gene bodies generally have low Xist coverage,their flanking regions are often marked by high Xist coverage suggestiveof a boundary that sequesters Xist and prevents it from entering escapeeloci (FIG. 6A, Mid1 shown)(Simon et al., 2013). Intriguingly, metageneanalysis revealed that PAR-TERRA was highly enriched at thetranscriptional start sites (TSS) of escapee genes, where the Xistboundary occurred (FIG. 6B). For example, Mid1 was marked by a sharpborder of Xist RNA beyond which Xist coverage dropped off dramatically(FIG. 6A). To determine how PAR-TERRA affected Xist spreading nearescapees, we carried out CHART-seq of Xist following knockdown ofPAR-TERRA for 6 hours. Scatterplot analysis of Xist coverages in 40-kbwindows showed that Xist binding changed very little for nearly all Xiloci (FIG. 6C, Pearson's r>0.90), with the notable exception of outlierscorresponding to some escapees (FIG. 6C, red dots). Upon PAR-TERRA KD,we observed decreased accumulation of Xist at the TSS and a shift toflanking regions (FIG. 6D). Taken together, these data suggest thatPAR-TERRA protects escapees from silencing by sequestering Xist at theTSS of escapees, thereby preventing the spread of Xist into upstreamregulatory regions and into the TSS regions of escapees.

Given that ˜90% of PAR-TERRA foci occurred next to but did not overlapthe Xist cloud (FIG. 6E; 8D)(Zhang et al., 2009), we considered thepossibility that PAR-TERRA might organize a privileged,transcription-permissive compartment next to the Xi. We noted that PARhas a higher colocalization rate with other escapees, such as Fix andJpx, than with genes subject to XCI, such as Hprt (FIG. 6F; P<0.0001).Depleting PAR-TERRA resulted in a statistically significant decrease incolocalization frequency (FIG. 6G; P<0.02, 0.001). These data areconsistent with the idea of a privileged compartment that isgeographically close to the pseudoautosomal region.

Gene ontology (GO) analysis was performed, restricted to differentiallyexpressed genes in TERRA KD (P adjusted value<0.05 in DEseq2 analysis).The top 10 enriched biological process terms are listed in Table 2, inthe upregulated gene set or in downregulated gene set in MEFs.

TABLE 2 Category Term Count PValue Benjamini MEF up-regulatedGOTERM_BP_FAT GO: 0007155~cell adhesion 25 9.28E−08 0.911919899GOTERM_BP_FAT GO: 0022610~biological adhesion 25 9.54E−06 0.006157583GOTERM_BP_FAT GO: 0030029~actin filament-based process 12 1.12E−040.047233272 GOTERM_BP_FAT GO: 0034330~cell fuction organization 61.35E−04 0.042637379 GOTERM_BP_FAT GO: 0030036~actin cytoskeletonorganization 11 2.94E−04 0.073349276 GOTERM_BP_FAT GO: 0034329~cellfunction assembly 5 2.95E−04 0.061751534 GOTERM_BP_FAT GO:0010810~regulation of cell-substrate adhesion 6 3.97E−04 0.070796403GOTERM_BP_FAT GO: 0034621~cellular macromolecular complex subunitorganization 13 5.34E−04 0.082874247 GOTERM_BP_FAT GO:0001944~vasculature development 13 8.40E−04 0.087939941 GOTERM_BP_FATGO: 0034622~cellular macromolecular complex assembly 12 6.75E−040.083770541 MEF down-regulated GOTERM_BP_FAT GO: 0007049~cell cycle 272.17E−08 2.99E−05 GOTERM_BP_FAT GO: 0051726~regulation of cell cycle 142.06E−06 0.001425534 GOTERM_BP_FAT GO: 0007348~regulation of mitoticcell cycle 10 2.42E−06 0.00111682 GOTERM_BP_FAT GO: 0008219~cell death21 3.42E−06 0.001182773 GOTERM_BP_FAT GO: 0012501~programmed cell death20 4.72E−06 0.001304329 GOTERM_BP_FAT GO: 0016265~death 21 4.86E−060.001120723 GOTERM_BP_FAT GO: 0006915~apoptosis 19 1.41E−05 0.002783689GOTERM_BP_FAT GO: 0051130~positive regulation of cellular componentorganization 10 1.74E−05 0.00300798 GOTERM_BP_FAT GO: 0033043~regulationof organelle organization 11 1.81E−05 0.002783466 GOTERM_BP_FAT GO:0022402~cell cycle process 17 2.36E−05 0.00325865

Example 6 TERRA Facilitates Homologous Sex Chromosome Pairing

In mammals, pairing between two homologous chromosomes rarely occursoutside of meiosis. An exception is the X-chromosome, which undergoestransient homologous interactions prior to the initiation of XCI in EScells (Bacher et al., 2006; Xu et al., 2006). Their transient pairingvia the noncoding Tsix and Xite loci is proposed to result in mutuallyexclusive selection of Xa and Xi and, thereby, to ensure upregulation ofXist RNA from a single ChrX (Xu et al., 2006). To date, only a fewregulatory factors have been identified, including a 15-kb “pairingcenter” from which the noncoding Tsix and Xite RNAs are produced (Xu etal., 2007). Tsix and Xite RNAs work together with the chromosomalarchitectural protein, CTCF, to establish the paired state (Kung et al.,2015). How the two X-chromosomes search and identify each other duringthe pairing process is not known.

Interestingly, PAR-TERRA ChIRT revealed strong PAR-TERRA binding sitesin Xite and Tsix (FIG. 7A). These peaks grabbed our attention becausethey occurred within the pairing center and were specific to ES cells.To test a role in pairing, we knocked down TERRA and measuredinterchromosomal distances in a 3D DNA FISH assay (Xu et al., 2006; Kunget al., 2015). We measured inter-allelic distances at the telomere, theX-inactivation center (Xic), and a distant locus, Hprt. Using Xic probesmapping to Tsix/Xist, we observed a left shift in the cumulativefrequency curve between days 0 and 4 of differentiation, indicating anincrease in the number of nuclei displaying inter-allelic distances of<0.1 nuclear diameters (ND) or <1 micron (FIG. 7B, 13A), a distanceimplying the occurrence of pairing, as previously determined (Bacher etal., 2006; Xu et al., 2006). By contrast, the number of such eventsincreased minimally between Hprt alleles. Curiously, however, whenmeasured between the telomeres (TeloX) or between PAR, there was also asignificant increase in allelic colocalization events. The degree ofleft shift indicated a very robust telomeric pairing (FIG. 7B).Telomeric pairing was X-specific and was not observed between homologousautosomal telomeres (FIG. 7C, Chr2 telomere shown). Moreover, the timewindow of telomeric pairing overlapped with that for Xic-Xic pairing.

This unexpected relationship raised the possibility that the X-telomeresmay be involved in somatic X-X pairing. Since the 1920's, the telomerehas been suspected to facilitate homology searching during meioticchromosome pairing, where a single telomeric “bouquet” (a clustering oftelomeric ends of all chromosomes) nucleates synapsis and enablessynaptic extension along the length of homologous pairs (Maguire, 1984;Rockmill and Roeder, 1998; Reig-Viader et al., 2013; Xiang et al.,2014). During male meiosis, Chr X and Y also pair in spite of theirlimited homology, and they do so via their pseudoautosomal region—theonly region of homology between the two sex chromosomes.

We explored the possibility that telomeric clustering could also mediatesex chromosome pairing in non-meiotic cells. First, given the occurrenceof meiotic X-Y pairing, we asked whether somatic telomeric pairingextends to the X and the Y. Indeed, during differentiation, theinter-PAR and inter-telomeric distances between Chr X and Y shifted tothe left, much in the same way as those observed for X-X telomericpairing (FIG. 7D, 13B). This colocalization of telomeres/PARs was alsotransient, occurring on day 4, but not on day 0 or 12. Serial RNA/DNAFISH showed that, while the paired telomeric signals of the sexchromosomes were very close (<1 μm between two dots) or overlapped (onedot), the rest of the two chromosomes were not paired, as visualized byChr X and Y painting probes (FIG. 7E). These data demonstrate thatsomatic telomeric pairing is not limited to the two femaleX-chromosomes. Transient homologous chromosome pairing also occursbetween Chr X and Y during differentiation of male ES cells.

We asked whether X-X and X-Y pairing might be controlled by PAR-TERRA incis. To examine telomeric pairing, we assessed pairing frequenciesbetween telomeres on d4 after treating cells with LNA gapmers to degradeTERRA RNA. Significantly, we observed a right shift of inter-telomeric(PAR-PAR) distances in TERRA KD cells relative to control cells thatwere administered a scrambled (Scr) LNA. These results demonstrated aloss of telomeric colocalization when TERRA RNA was depleted (FIG. 7F).Telomeric X-X pairing in female cells and X-Y pairing in male cells wereboth affected. Therefore, TERRA RNA is required for somatic telomericpairing of sex chromosomes. Given the presence of PAR-TERRA bindingsites at the pairing center, we investigated whether Xic-Xic pairing infemale cells might also be affected by the loss of TERRA. We treateddifferentiating female ES cells with TERRA LNAs and measured inter Xicdistances at day 4. Relative to cells treated with the control Scr LNA,there was a significant right shift of colocalization frequenciesbetween the Xic alleles after TERRA KD (FIG. 7G), indicating a loss ofpairing. Thus, both types of trans-interactions—telomeric pairing andXic-Xic pairing—require the function of TERRA.

These data lead to the notion that PAR-TERRA RNA could facilitatehomologous interchromosomal interactions by inducing co-clustering ofcrucial pairing sites. Because the pairing center (FIG. 7A) and thepseudoautosomal region (FIG. 2H) are both major hotspots of PAR-TERRAbinding, we examined the possibility that intra-chromosomalcis-interactions between the Xic and telomere could bring the Xic to thejuxta-telomeric compartment, which in turn would bring the two Xic's inclose proximity due to the action of telomeric pairing. Indeed, DNA FISHusing Xic and sub-telomeric (PAR) probes showed that the Xic andtelomere frequently colocalized on day 4 (FIG. 7H). This co-clusteringdepended on TERRA RNA, as knocking down TERRA abolished theXic-telomeric interactions (FIG. 7H). We conclude that both (i)cis-interactions between the Xic and the telomere and (ii)trans-interactions between two telomeres require TERRA RNA. We proposethat TERRA RNA tethers the ends of sex chromosomes to facilitateinter-chromosomal interactions between the Xic's (FIG. 7I).

REFERENCES

Azzalin, C. M., and Lingner, J. (2015). Telomere functions grounding onTERRA firma. Trends Cell Biol 25, 29-36

Azzalin, C. M., Reichenbach, P., Khoriauli, L., Giulotto, E., andLingner, J. (2007). Telomeric Repeat Containing RNA and RNA SurveillanceFactors at Mammalian Chromosome Ends. Science 318, 798-801

Bacher, C. P., Guggiari, M., Brors, B., Augui, S., Clerc, P., Avner, P.,Eils, R., and Heard, E. (2006). Transient colocalization ofX-inactivation centres accompanies the initiation of X inactivation.Nature cell biology 8, 293-299

Balk, B., Maicher, A., Dees, M., Klermund, J., Luke-Glaser, S., Bender,K., and Luke, B. (2013). Telomeric RNA-DNA hybrids affecttelomere-length dynamics and senescence. Nat Struct Mol Biol 20,1199-1205

Berletch, J. B., Yang, F., and Disteche, C. M. (2010). Escape from Xinactivation in mice and humans. Genome biology 11, 213

Berletch, J. B., Yang, F., Xu, J., Carrel, L., and Disteche, C. M.(2011). Genes that escape from X inactivation. Human genetics 130,237-245

Bernardes de Jesus, B., and Blasco, M. A. (2013). Telomerase at theintersection of cancer and aging. Trends in genetics: TIG 29, 513-520

Blackburn, E. H., Greider, C. W., and Szostak, J. W. (2006). Telomeresand telomerase: the path from maize, Tetrahymena and yeast to humancancer and aging. Nature medicine 12, 1133-1138

Brown, C. J., Hendrich, B. D., Rupert, J. L., Lafreniere, R. G., Xing,Y., Lawrence, J., and Willard, H. F. (1992). The human XIST gene:analysis of a 17 kb inactive X-specific RNA that contains conservedrepeats and is highly localized within the nucleus. Cell 71, 527-542

Carrel, L., and Willard, H. F. (2005). X-inactivation profile revealsextensive variability in X-linked gene expression in females. Nature434, 400-404

Chu, C., Qu, K., Zhong, F. L., Artandi, S. E., and Chang, H. Y. (2011).Genomic maps of long noncoding RNA occupancy reveal principles ofRNA-chromatin interactions. Molecular cell 44, 667-678

de Silanes, I. L., Grana, O., De Bonis, M. L., Dominguez, O., Pisano, D.G., and Blasco, M. A. (2014). Identification of TERRA locus unveils atelomere protection role through association to nearly all chromosomes.Nat Commun 5, 4723

Deng, X., Berletch, J. B., Nguyen, D. K., and Disteche, C. M. (2014). Xchromosome regulation: diverse patterns in development, tissues anddisease. Nat Rev Genet 15, 367-378

Deng, Z., Norseen, J., Wiedmer, A., Riethman, H., and Lieberman, P. M.(2009). TERRA RNA binding to TRF2 facilitates heterochromatin formationand ORC recruitment at telomeres. Molecular cell 35, 403-413

Disteche, C. M. (2012). Dosage compensation of the sex chromosomes.Annual review of genetics 46, 537-560

Dixon, J. R., Selvaraj, S., Yue, F., Kim, A., Li, Y., Shen, Y., Hu, M.,Liu, J. S., and Ren, B. (2012). Topological domains in mammalian genomesidentified by analysis of chromatin interactions. Nature 485, 376-380

Doksani, Y., and de Lange, T. (2014). The role of double-strand breakrepair pathways at functional and dysfunctional telomeres. Cold SpringHarbor perspectives in biology 6, a016576

Filippova, G. N., Cheng, M. K., Moore, J. M., Truong, J. P., Hu, Y. J.,Nguyen, D. K., Tsuchiya, K. D., and Disteche, C. M. (2005). Boundariesbetween chromosomal domains of X inactivation and escape bind CTCF andlack CpG methylation during early development. Dev Cell 8, 31-42

Heinz, S., Benner, C., Spann, N., Bertolino, E., Lin, Y. C., Laslo, P.,Cheng, J. X., Murre, C., Singh, H., and Glass, C. K. (2010). Simplecombinations of lineage-determining transcription factors primecis-regulatory elements required for macrophage and B cell identities.Molecular cell 38, 576-589

Horvath, L. M., Li, N., and Carrel, L. (2013). Deletion of anX-inactivation boundary disrupts adjacent gene silencing. PLoS genetics9, e1003952

Kharchenko, P. V., Tolstorukov, M. Y., and Park, P. J. (2008). Designand analysis of ChIP-seq experiments for DNA-binding proteins. Naturebiotechnology 26, 1351-1359

Kung, J. T., Kesner, B., An, J. Y., Ahn, J. Y., Cifuentes-Rojas, C.,Colognori, D., Jeon, Y., Szanto, A., Del Rosario, B. C., Pinter, S. F.,et al. (2015). Locus-Specific Targeting to the X Chromosome Revealed bythe RNA Interactome of CTCF. Molecular cell 57, 361-375

Le, P. N., Maranon, D. G., Altina, N. H., Battaglia, C. L., and Bailey,S. M. (2013). TERRA, hnRNP A1, and DNA-PKcs Interactions at HumanTelomeres. Frontiers in oncology 3, 91

Lee, J. T. (2011). Gracefully ageing at 50, X-chromosome inactivationbecomes a paradigm for RNA and chromatin control. Nat Rev Mol Cell Biol12, 815-826

Lee, J. T., Davidow, L. S., and Warshawsky, D. (1999). Tsix, a geneantisense to Xist at the X-inactivation centre. Nat Genet 21, 400-404

Lingner, J., Hughes, T. R., Shevchenko, A., Mann, M., Lundblad, V., andCech, T. R. (1997). Reverse transcriptase motifs in the catalyticsubunit of telomerase. Science 276, 561-567

Lopes, A. M., Arnold-Croop, S. E., Amorim, A., and Carrel, L. (2011).Clustered transcripts that escape X inactivation at mouse XqD. Mammaliangenome: official journal of the International Mammalian Genome Society22, 572-582

Luke, B., Panza, A., Redon, S., Iglesias, N., Li, Z., and Lingner, J.(2008). The Rat 1p 5′ to 3′ exonuclease degrades telomericrepeat-containing RNA and promotes telomere elongation in Saccharomycescerevisiae. Molecular cell 32, 465-477

Maguire, M. P. (1984). The mechanism of meiotic homologue pairing.Journal of theoretical biology 106, 605-615

Maicher, A., Kastner, L., Dees, M., and Luke, B. (2012). Deregulatedtelomere transcription causes replication-dependent telomere shorteningand promotes cellular senescence. Nucleic Acids Res 40, 6649-6659

Merkenschlager, M., and Odom, D. T. (2013). CTCF and cohesin: linkinggene regulatory elements with their targets. Cell 152, 1285-1297

Penny, G. D., Kay, G. F., Sheardown, S. A., Rastan, S., and Brockdorff,N. (1996). Requirement for Xist in X chromosome inactivation. Nature379, 131-137

Pfeiffer, V., Crittin, J., Grolimund, L., and Lingner, J. (2013). TheTHO complex component Thp2 counteracts telomeric R-loops and telomereshortening. EMBO J 32, 2861-2871

Pfeiffer, V., and Lingner, J. (2012). TERRA promotes telomere shorteningthrough exonuclease 1-mediated resection of chromosome ends. PLoSgenetics 8, e1002747

Pinter, S. F., Sadreyev, Yildirim, E., Jeon, Y., Ohsumi, T. K.,Borowsky, M., and Lee, J. T. (2012). Spreading of X chromosomeinactivation via a hierarchy of defined Polycomb stations. Genomeresearch 22, 1864-1876

Redon, S., Reichenbach, P., and Lingner, J. (2010). The non-coding RNATERRA is a natural ligand and direct inhibitor of human telomerase.Nucleic Acids Res 38, 5797-5806

Redon, S., Zemp, I., and Lingner, J. (2013). A three-state model for theregulation of telomerase by TERRA and hnRNPAl. Nucleic Acids Res 41,9117-9128

Reig-Viader, R., Brieno-Enriquez, M. A., Khouriauli, L., Toran, N.,Cabero, L., Giulotto, E., Garcia-Caldes, M., and Ruiz-Herrera, A.(2013). Telomeric repeat-containing RNA and telomerase in human fetaloocytes. Hum Reprod 28, 414-422

Reig-Viader, R., Vila-Cejudo, M., Vitelli, V., Busca, R., Sabate, M.,Giulotto, E., Caldes, M. G., and Ruiz-Herrera, A. (2014). Telomericrepeat-containing RNA (TERRA) and telomerase are components of telomeresduring mammalian gametogenesis. Biol Reprod 90, 103

Rockmill, B., and Roeder, G. S. (1998). Telomere-mediated chromosomepairing during meiosis in budding yeast. Genes & development 12,2574-2586

Sandell, L. L., Gottschling, D. E., and Zakian, V. A. (1994).Transcription of a yeast telomere alleviates telomere position effectwithout affecting chromosome stability. Proceedings of the NationalAcademy of Sciences of the United States of America 91, 12061-12065

Schoeftner, S., and Blasco, M. A. (2007). Developmentally regulatedtranscription of mammalian telomeres by DNA-dependent RNA polymerase II.Nature cell biology 10, 228-236

Schoeftner, S., and Blasco, M. A. (2008). Developmentally regulatedtranscription of mammalian telomeres by DNA-dependent RNA polymerase II.Nature cell biology 10, 228-236

Sfeir, A., and de Lange, T. (2012). Removal of shelterin reveals thetelomere end-protection problem. Science 336, 593-597

Shin, H., Liu, T., Manrai, A. K., and Liu, X. S. (2009). CEAS:cis-regulatory element annotation system. Bioinformatics 25, 2605-2606

Simon, M. D., Pinter, S. F., Fang, R., Sarma, K., Rutenberg-Schoenberg,M., Bowman, S. K., Kesner, B. A., Maier, V. K., Kingston, R. E., andLee, J. T. (2013). High-resolution Xist binding maps reveal two-stepspreading during X-chromosome inactivation. Nature 504, 465-469

Simon, M. D., Wang, C. I., Kharchenko, P. V., West, J. A., Chapman, B.A., Alekseyenko, A. A., Borowsky, M. L., Kuroda, M. I., and Kingston, R.E. (2011). The genomic binding sites of a noncoding RNA. Proceedings ofthe National Academy of Sciences of the United States of America 108,20497-20502

Soriano, P., Keitges, E. A., Schorderet, D. F., Harbers, K., Gartler, S.M., and Jaenisch, R. (1987). High rate of recombination and doublecrossovers in the mouse pseudoautosomal region during male meiosis.Proceedings of the National Academy of Sciences of the United States ofAmerica 84, 7218-7220

Starmer, J., and Magnuson, T. (2009). A new model for random Xchromosome inactivation. Development 136, 1-10

Sun, S., Del Rosario, B. C., Szanto, A., Ogawa, Y., Jeon, Y., and Lee,J. T. (2013). Jpx RNA activates Xist by evicting CTCF. Cell 153,1537-1551

Trapnell, C., Hendrickson, D. G., Sauvageau, M., Goff, L., Rinn, J. L.,and Pachter, L. (2013). Differential analysis of gene regulation attranscript resolution with RNA-seq. Nature biotechnology 31, 46-53

Wang, C., Zhao, L., and Lu, S. (2015). Role of TERRA in the Regulationof Telomere Length. Int J Biol Sci 11, 316-323

Wutz, A. (2011). Gene silencing in X-chromosome inactivation: advancesin understanding facultative heterochromatin formation. Nat Rev Genet12, 542-553

Xiang, Y., Miller, D. E., Ross, E. J., Sanchez Alvarado, A., and Hawley,R. S. (2014). Synaptonemal complex extension from clustered telomeresmediates full-length chromosome pairing in Schmidtea mediterranea.Proceedings of the National Academy of Sciences of the United States ofAmerica 111, E5159-5168

Xu, N., Donohoe, M. E., Silva, S. S., and Lee, J. T. (2007). Evidencethat homologous X-chromosome pairing requires transcription and Ctcfprotein. Nat Genet 39, 1390-1396

Xu, N., Tsai, C. L., and Lee, J. T. (2006). Transient homologouschromosome pairing marks the onset of X inactivation. Science 311,1149-1152

Yang, F., Babak, T., Shendure, J., and Disteche, C. M. (2010). Globalsurvey of escape from X inactivation by RNA-sequencing in mouse. Genomeresearch 20, 614-622

Yu, T. Y., Kao, Y. W., and Lin, J. J. (2014). Telomeric transcriptsstimulate telomere recombination to suppress senescence in cells lackingtelomerase. Proceedings of the National Academy of Sciences of theUnited States of America 111, 3377-3382

Zhang, L. -F., Ogawa, Y., Ahn, J. Y., Namekawa, S. H., Silva, S. S., andLee, J. T. (2009). Telomeric RNAs Mark Sex Chromosomes in Stem Cells.Genetics 182, 685

Zhang, Y., Liu, T., Meyer, C. A., Eeckhoute, J., Johnson, D. S.,Bernstein, B. E., Nusbaum, C., Myers, R. M., Brown, M., Li, W., et al.(2008). Model-based analysis of ChIP-Seq (MACS). Genome biology 9, R137

Zhao, J., Sun, B. K., Erwin, J. A., Song, J. J., and Lee, J. T. (2008).Polycomb proteins targeted by a short repeat RNA to the mouse Xchromosome. Science 322, 750-756

OTHER EMBODIMENTS

It is to be understood that while the invention has been described inconjunction with the detailed description thereof, the foregoingdescription is intended to illustrate and not limit the scope of theinvention, which is defined by the scope of the appended claims. Otheraspects, advantages, and modifications are within the scope of thefollowing claims.

1. A composition comprising an isolated inhibitory nucleic acidtargeting PAR-TERRA, wherein the inhibitory nucleic acid is modified. 2.A method of decreasing expression of an Xi escapee gene, or decreasingexpression of Xa genes, in a cell, the method comprising administeringto the cell an inhibitory nucleic acid targeting PAR-TERRA.
 3. Themethod of claim 2, comprising decreasing expression of Xa genes in acell.
 4. The method of claim 3, wherein the cell is in subject who has adisorder of sex chromosome aneuploidy associated with a supernumerary Xchromosome, the method comprising administering to the subject aninhibitory nucleic acid targeting PAR-TERRA.
 5. A method of decreasingexpression of autosomal genes in a cell, the method comprisingadministering to the cell an inhibitory nucleic acid targeting PAR-TERRAor an autosome-specific TERRA, optionally TERRA species originatingwithin the subtelomeric region of an autosome and comprisingautosome-specific 5′ sequences, optionally wherein the inhibitorynucleic acid is modified.
 6. The method of claim 5, wherein theinhibitory nucleic acid targets Chr4-specific TERRA.
 7. The method ofclaim 6, wherein expression of FRG1, FRG2, DUX4, and the long noncodingRNAs of forward and reverse orientations from the macrosatellite repeat,D4Z4, is decreased.
 8. The method of claim 6, wherein the cell is fromor in a subjection who has facioscapulohumeral muscular dystrophy(FSHD).
 9. (canceled)
 10. The method of claim 4, wherein the subject has46,XY, 47,XXY, 48,XXYY, 48,XXXY, 47,XXX, 48,XXXX or 49,XXXXX aneuploidy.11. The method of claim 2, wherein the cell is from a subject who has46,XY, 47,XXY, 48,XXYY, 48,XXXY, 47,XXX, 48,XXXX or 49,XXXXX aneuploidy.12. A method of decreasing expression of X-linked or autosomal growthcontrol or apoptosis genes in a cell, the method comprisingadministering to the cell an inhibitory nucleic acid targetingPAR-TERRA, PAR, or TERRA, optionally wherein the inhibitory nucleic acidis modified.
 13. The method of claim 1, wherein the inhibitory nucleicacid does not comprise three or more consecutive guanosine nucleotidesor does not comprise four or more consecutive guanosine nucleotides. 14.The method of claim 1, wherein the inhibitory nucleic acid is 8 to 30nucleotides in length.
 15. The method of claim 1, wherein at least onenucleotide of the inhibitory nucleic acid is a nucleotide analogue. 16.The method of claim 1, wherein at least one nucleotide of the inhibitorynucleic acid comprises a 2′ O-methyl, e.g., wherein each nucleotide ofthe inhibitory nucleic acid comprises a 2′ O-methyl.
 17. The method ofclaim 1, wherein the inhibitory nucleic acid comprises at least oneribonucleotide, at least one deoxyribonucleotide, or at least onebridged nucleotide.
 18. The method of claim 17, wherein the bridgednucleotide is a LNA nucleotide, a cEt nucleotide or a ENA modifiednucleotide.
 19. The method of claim 1, wherein each nucleotide of theinhibitory nucleic acid is a LNA nucleotide.
 20. The method of claim 19,wherein one or more of the nucleotides of the inhibitory nucleic acidcomprise 2′-fluoro-deoxyribonucleotides and/or 2′-O-methyl nucleotides.21. The method of claim 1, wherein one or more of the nucleotides of theinhibitory nucleic acid comprise one of both of ENA nucleotide analoguesor LNA nucleotides.
 22. The method of claim 1, wherein the nucleotidesof the inhibitory nucleic acid comprise comprising phosphorothioateinternucleotide linkages between at least two nucleotides, or betweenall nucleotides.
 23. The method of claim 1, wherein the inhibitorynucleic acid is a gapmer or a mixmer.